naphthoquinones and plumbagin

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

The genus Plumbago (family Plumbaginaceae), commonly known as leadwort, is a sub-tropical shrub that produces secondary metabolite plumbagin, which is employed by pharmaceutical companies and in clinical research. Plumbagin is a potent pharmaceutical because of its anti-microbial, anti-malarial, antifungal, anti-inflammatory, anti-carcinogenic, anti-fertility, anti-plasmodium, antioxidant, anti-diabetic, and other effects. This review documents the biotechnological innovations used to produce plumbagin. The use of modern biotechnological techniques can lead to a variety of benefits, including better yield, increased extraction efficiency, mass production of plantlets, genetic stability, increased biomass, and more. Large-scale in vitro propagation is necessary to minimize over-exploitation of the natural population and allow the use of various biotechnological techniques to improve the plant species and secondary metabolite production. During in vitro culture, optimum conditions are requisites for explant inoculation and plant regeneration. In this review, we provide information on various aspects of plumbagin, depicting its structure, biosynthesis, and biotechnological aspects (both conventional and advanced) along with the future prospects. KEY POINTS: • Critical assessment on in vitro biotechnology in Plumbago species • In vitro propagation of Plumbago and elicitation of plumbagin • Biosynthesis and sustainable production of plumbagin.

Topics: Biotechnology; Naphthoquinones; Pharmaceutical Preparations; Plumbaginaceae

Bone metabolism is essential for maintaining bone mineral density and bone strength through a balance between bone formation and bone resorption. Bone formation is associated with osteoblast activity whereas bone resorption is linked to osteoclast differentiation. Osteoblast progenitors give rise to the formation of mature osteoblasts whereas monocytes are the precursors for multi-nucleated osteoclasts. Chronic inflammation, auto-inflammation, hormonal changes or adiposity have the potential to disturb the balance between bone formation and bone loss. Several plant-derived components are described to modulate bone metabolism and alleviate osteoporosis by enhancing bone formation and inhibiting bone resorption. The plant-derived naphthoquinone plumbagin is a bioactive compound that can be isolated from the roots of the

Topics: Bone Resorption; Cell Differentiation; Humans; Inflammation; Naphthoquinones; Osteoblasts; Osteoclasts; Phytochemicals

Plumbagin has gained a lot of attention in research due to its various therapeutic actions. It is a secondary metabolite obtained from different plant families, such as Plumbaginaceae, Droseraceae, and Ebenceae. Various studies on plumbagin have revealed that it is a natural gift for mankind in treating chronic diseases, like cancer, diabetes, malaria, bacterial infection, and controlling cardiovascular disease. However, there are several challenges in developing plumbagin as a therapeutic agent. The first and foremost is its limited solubility and oral bioavailability. The second limitation is its toxicity. Plumbagin has a narrow therapeutic window, and literature reveals that the compound has moderate toxicity in animals. However, data are insufficient to prove that plumbagin is unsafe for humans. Despite the many therapeutic benefits of plumbagin, it remains unexploited for mankind. Thus, a systematic review of its toxicity, pharmacology, and safety is required to be performed. This review work signifies the depth of therapeutic applications proven via research, its different modes of isolation and separation of chemical constituents, and its modification. A thorough review of promising therapeutic targets via docking studies is also presented. Different methods used to quantify plumbagin from the plant are reviewed. An overview of attempts to design novel formulations which could enhance its bioavailability is also presented. The review paper will help the scientist to exploit the drug to its optimum, which will help to overcome the challenges faced during its design and developmental stages.

Topics: Animals; Humans; Naphthoquinones; Neoplasms; Plumbaginaceae; Research

Recently, natural products have been became the center of attraction for the scientific society and exploration of their biologically abilities is proceeding continuously. In search for novel antiparasitic agents with an objective of protecting humans from parasitic infections, the present work was focused on naphthoquinones possessing antiparasitic activity. Among naphthoquinones, plumbagin is one of the secondary metabolites exhibiting diverse biological properties such as antibacterial, antimalarial, antiinflammatory, insecticidal and antiparasitic. Plumbagin is reported to have antischistosomiasis, anti-haemonchosis, anti-fascioliasis, antiotoacariasis, anti-leishmaniasis, antimalaria, antiallergic and anthelmintic activities. Besides, various methods of extraction of plumbagin from different methods, their effectiveness against different parasites, and the structure-activity relationship reported by different researchers. This work highlight on recent advancements in the phytochemistry of plumbagin, studies associated with various biological activities. The structure-activity relationship studies have also been summarized. To conclude, present review could be beneficial for the scientific community to get better insight into medicinal research of plumbagin and may provide a new horizon for the rational design of plumbagin based compounds.

Topics: Anti-Infective Agents; Antiparasitic Agents; Humans; Naphthoquinones; Parasitic Diseases

Cancer is a deadly disease, which has significantly increased in both developed and developing nations. Treatment of cancer utilizing radiotherapy or chemotherapy actuates a few issues which incorporate spewing, sickness, unpalatable reactions, and so forth. In this specific situation, an alternative drug source, which can effectively treat cancer is of prime importance. Products that are obtained from plant sources are utilized for the treatment of various diseases due to their non-harmful nature. Medicinal plants contain different bioactive compounds, which possess an important role in the prevention of different diseases such as cancer. Plumbagin is a bioactive compound, which is mainly present in Plumbaginaceae family and has been explored for its anticancer activity. Plumbagin basically inactivates the Akt/NF-kB, MMP-9 and VEGF pathways that are essential for cancer cell development. Therefore, it is important to review the role of plumbagin in different cancer cells in order to find an alternative drug to overcome this disease. The present review provides a summary of anticancer activity of plumbagin in various cancers and its mode of action.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle Checkpoints; Humans; Naphthoquinones; Neoplasms; NF-kappa B; Plumbaginaceae; Signal Transduction

Plumbagin (PLB), a natural naphthoquinone constituent isolated from the roots of the medicinal plant

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; DNA Breaks, Double-Stranded; DNA Damage; Drug Screening Assays, Antitumor; Glutathione; Humans; Inhibitory Concentration 50; Liposomes; Mice; Mice, Inbred C57BL; Naphthoquinones; Neoplasm Metastasis; Neoplasms; Oxidants; Oxygen; Plant Extracts; Plants, Medicinal; Proteasome Inhibitors; Reactive Oxygen Species; Superoxides

Plumbagin is a naphthoquinone derived yellow crystalline phytochemical. Plumbagin has a wide range of biological effects including cytotoxicity against cancer cells both in vitro and in vivo. Due to the pleiotropic nature of plumbagin, it shows the anticancer effect by targeting several molecular mechanisms including apoptosis and autophagic pathways, cell cycle arrest, anti-angiogenic pathways, anti-invasion and anti-metastasis pathways. Among many signaling pathways the key regulatory genes regulated by plumbagin are NF-kβ, STAT3, and AKT, etc. Plumbagin is also a potent inducer of ROS, suppressor of cellular glutathione, and causes DNA strand break by oxidative DNA base damages. In vivo studies suggested that plumbagin significantly reduces the tumor weight and volume in dose-dependent manner without any side effects in tested model organisms. Another exciting aspect of plumbagin is the ability to re-sensitize the chemo and radioresistant cancer cells when used in combination or alone. Nano encapsulation of plumbagin overcomes the poor water solubility and bioavailability obstacles, enhancing the pharmaceutical relevance with better therapeutic efficacy. Moreover, plumbagin can be introduced as a future phytotherapeutic anticancer drug after fully satisfied preclinical and clinical trials.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Humans; Male; Naphthoquinones; Neoplasms; Reactive Oxygen Species; Signal Transduction

Plumbagin (PLB), a member of the quinine family, mainly found in the plant Plumbago zeylanica Linn., potentially exhibit anticancer, anti-inflammatory, anti-oxidant, antifungal, neuroprotective, hypolipidemic and antibacterial activities. However, it has been well known that the application of PLB was limited owing to its water insolubility, instability and poor bioavailability. For decades, many attempts have been made to compensate for these disadvantages with the development of improved delivery platforms as the feasible approaches. This review aims to describe the various studies supporting the biopharmaceutical aspects of PLB. In addition, it includes a section devoted to discussing the challenges associated with the drug and strategies to improve the properties of PLB such as solubility, stability and bioavailability. Also, this paper summarizes the recent works on the design and development of novel delivery systems of PLB such as liposomes, niosomes, microsphares, nanoparticles, micelles, complexization, metal nanoparticles, crystals modification, etc., with the goal of harnessing the true difficulties of this multifunctional agent in the clinical arena.

Topics: Drug Carriers; Drug Discovery; Naphthoquinones

Increased demand for compounds that are derived from natural source are gaining more and more importance. Plumbagin is a plant naphthoquinone which is present in several families, including Iridaceae, Plumbaginaceae, Ebenceae, Drosophyllaceae, Nepenthaceae and Droseraceae. Plumbagin possesses high therapeutic efficacy and minimal side effects. It has various pharmaceutical activities which include anticancer, antibacterial, anti-inflammatory, antioxidant, antifungal, neuroprotective and hypolipidemic activities. In natural habitat, production of plumbagin is low due to species variations and environmental changes, considering importance of this bioactive compound, alternative techniques for its enhanced production needs to be devised. In the present review, various production techniques and scale-up strategies for plumbagin production are discussed.. Aim of this review is to provide an insight into the chemistry of plumbagin, its pharmaceutical activities, perspective of cell suspension culture, root culture, hairy root culture and scale up strategies for its production.. All the data compiled and presented here were obtained from various E-resources like Pubmed, Science Direct, and Google Scholar up to February 2018.. This review comprises isolation, extraction and quantification method for plumbagin, its pharmaceutical activities, various tissue culture production techniques and scale-up strategies for enhanced production.. Plumbagin is an important phytocompound which shows potential towards treatments of various diseases. Demand for the production of plumbagin continuously increasing worldwide due to its pharmacological properties. To fulfil commercial demand of plumbagin alternative technologies need to be investigated. Biotechnological approaches like cell suspension culture, root suspension culture and hairy root culture are alternative techniques for plumbagin production. These techniques provide continuous supply of bioactive compounds. However, research on various aspects of tissue culture production techniques is in preparatory stage and requires culture and process optimization for development of a commercially practical process.

Topics: Biotechnology; Cell Culture Techniques; Naphthoquinones; Plant Roots; Plumbaginaceae

It has been shown that plumbagin, a bioactive naphthoquinone isolated from three major plant families viz. Plumbaginaceae, Ebenceae and Droseraceae, definitively exhibits anticancer potential in diverse cancer cells both in vitro and in vivo. Plumbagin shows antineoplastic effects via multi-channel molecular mechanisms, including the induction of apoptosis and autophagy, the disruption of the cell cycle, the inhibition of invasion and metastasis, and anti-angiogenesis. Plumbagin inhibits the growth of cancer cells mainly through the modulation of the signals of PI3K/Akt/mTOR, AMPK, Ras, and so on. The pharmaceutical applications of plumbagin combined with nanocarriers to achieve better therapeutic efficiency are discussed in this review Among them, liposomes, nanoparticles, microspheres, micelles, and nisosomes are used in cancer treatment. The anticancer study of plumbagin in vivo is also summarized in this review. On the whole, we aim to review the research progress of plumbagin both in pharmacological and pharmaceutical filed, which may provide some reference for further research of plumbagin.

Topics: AMP-Activated Protein Kinases; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Droseraceae; Ebenaceae; Genes, ras; Humans; In Vitro Techniques; Mice; Naphthoquinones; Neoplasms; Phosphatidylinositol 3-Kinase; Phytotherapy; Plumbaginaceae; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a plant-derived naphthoquinones obtained mainly from three families, including Plumbaginaceae, Droseraceae, and Ebenaceae. Plumbagin has exhibited its potential therapeutic benefits on numerous chronic diseases, i.e., breast cancer, non-small cell lung cancer, melanoma, ovarian, squamous cell carcinomas, pancreatic cancer, and prostate cancer. In addition, its anti-inflammatory and antimicrobial activities as well as control of diabetes and cardiovascular diseases have been reported. Thus, plumbagin is a promising agent for development as a new drug for the treatment or control of chronic diseases. Studies on controlled drug release or drug delivery systems have been involved for improvement of its therapeutic efficacy as well as for the reduction of its toxicity. However, most of the recent research information is from in vitro and in vivo studies. Further clinical studies are therefore required for its developments and applications as a novel drug used to treat chronic diseases.

Topics: Animals; Anti-Infective Agents; Anti-Inflammatory Agents; Antineoplastic Agents, Phytogenic; Cardiovascular Agents; Chronic Disease; Drug Discovery; Humans; Hypoglycemic Agents; Molecular Structure; Naphthoquinones; Phytotherapy; Plants, Medicinal; Signal Transduction

Recently, a wide range of food-derived phytochemical compounds and their synthetic derivatives have been proposed for cancer treatment. Unfortunately, data available in related literature focus on the anti-cancer properties of compounds derived from edible plants, while very little is known about those derived from non-edible plants. And thus, the underlying mechanisms of their anti-cancer effects are yet to be elucidated. This review collates the available data on the anti-cancer activities of six phytochemical-derived compounds from edible and non-edible plants, i.e. rottlerin, berbamine, sparstolonin B, sulforaphane, plumbagin and 6-shogaol. These compounds are used as bioactive markers for cytotoxicity against tumors. As such, understanding their mode of action will provide the rationale for the combination strategies of these compounds with other drugs in the battle against cancer.

Topics: Acetophenones; Antineoplastic Agents, Phytogenic; Benzopyrans; Benzylisoquinolines; Catechols; Heterocyclic Compounds, 4 or More Rings; Humans; Isothiocyanates; Naphthoquinones; Neoplasms; Phytotherapy; Plant Extracts; Signal Transduction; Sulfoxides

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

The genus Diospyros is one of the most important sources of bioactive compounds, exclusively 1,4-naphthoquinones. The following information is an attempt to cover the developments in the biology and phytochemistry of 1,4-naphthoquinones isolated from this genus, as well as the studies done and the suggested mechanisms regarding their activities. During the past 60 years, many of these agents have been isolated from Diospyros L. Twelve considerable bioactive structures are reported in this review. The basic 1,4-naphthoquinone skeletons, on which a large number of studies have been done, are plumbagin and diospyrin. Today, the potential for development of leads from 1,4-naphthoquinones obtained from Diospyros L. is growing dramatically, mainly in the area of anticancer and antibacterial investigations. The data prepared and described here are intended to be served as a reference tool to the natural products and chemistry specialists in order to expand the rational drug design.

Topics: Anti-Bacterial Agents; Antineoplastic Agents, Phytogenic; Diospyros; Naphthoquinones

Plumbagin is one of the simplest plant secondary metabolite of three major phylogenic families viz. Plumbaginaceae, Droseraceae, and Ebenceae, and exhibits highly potent biological activities, including antioxidant, antiinflammatory, anticancer, antibacterial, and antifungal activities. Recent investigations indicate that these activities arise mainly out of its ability to undergo redox cycling, generating reactive oxygen species and chelating trace metals in biological system. The compound is endowed with a property to inhibit the drug efflux mechanism in drug-resistant bacteria, thereby allowing intracellular accumulation of the potent drug molecules. An interesting bioactivity exhibited by this compound is the elimination of stringent, conjugative, multidrug-resistant plasmids from several bacterial strains including opportunistic bacteria, such as Acinetobacter baumannii. Moreover, plumbagin effectively induces apoptosis and causes cell cycle arrest, which is, in part, due to the inactivation of NF-κB in cancer cells. Therefore, it has been suggested that designing "hybrid drug molecules" of plumbagin by combining it with other appropriate anticancer agents may lead to the generation of novel and potent anticancer drugs with pleiotropic action against human cancers. This comprehensive review is an attempt to understand the chemistry of plumbagin and catalog its biological activities reported to date.

Topics: Animals; Biological Availability; Electrochemical Techniques; Humans; Metals; Naphthoquinones; Plants, Medicinal; Spectrum Analysis

Plumbagin and other naphthoquinone derivatives from the Plumbago zeylanica L. (Plumbaginaceae) are known for their anticancer and other medicinal properties. Previous reports suggest that 3-methyl-1,8-naphthalene-diol is an intermediate of the plumbagin biosynthetic pathway and is synthesized from hexaketide backbone; a reaction catalyzed by type III polyketide synthase (PKS) along with certain accessory enzymes. Our earlier transcriptomic and metabolomic studies suggest that along with PKS, putative cyclase and aldo-keto reductase might be involved in the formation of 3-methyl-1,8-naphthalene-diol. The present study probed young leaf transcriptome and identified cyclase and aldo-keto reductase like transcripts that might be involved in the intramolecular aldol condensation of hexaketide intermediate and decarboxylation, carbonyl reduction and hydroxyl elimination of keto or enol forms of hexaketide intermediates respectively. Moreover, sequence alignment of identified cyclase1 possesses signature β-α-β-β-α-α-β topology, which belongs to the dimeric α + β barrel (DABB) protein family and is involved in the C2-C11 and C4-C9 intramolecular aldol condensation of hexaketide intermediates. Along with cyclase1, we further identified and characterized P. zeylanica specific aldo-keto reductase1 (AKR1) which is a novel member of the aldo-keto reductase (AKR) multi-gene family that possesses the conserved Asp60, Tyr65, Lys91, and His132 residues and is proposed to be involved in the C1 decarboxylation, C3 carbonyl reduction and C7 hydroxyl elimination of keto or enol form of hexaketide intermediate to form 3-methyl-1,8-naphthalene-diol. Further, the functional characterization using the artificial microRNA mediated transient silencing approach confirmed the involvement of cyclase1 and AKR1 in the plumbagin biosynthetic pathway. This is the first study reporting the identification and functional characterization of cyclase1 and AKR1 genes involved in the plumbagin biosynthetic pathway and general plant polyketide biosynthesis.

Topics: Aldo-Keto Reductases; Biosynthetic Pathways; MicroRNAs; Naphthoquinones

Plumbagin is a natural compound known to impede growth of cancerous cells. However, anti-cervical cancer effects of plumbagin and its underlying molecular mechanism still remains elusive. In this study, plumbagin reduced the viability of CaSki cells in a concentration dependent manner and suppressed their colony formation potential. It led to G2/M phase arrest with downregulation of E2F1 and upregulation of p21. Plumbagin reduced mitochondrial membrane potential and concomitantly increased the percentage of apoptotic cells as revealed by annexin V-propidium iodide staining. Real Time PCR and western blotting confirmed that plumbagin induced apoptosis by reducing the expression of pAkt, procaspase 9 and full-length PARP. Furthermore, scratch assay showed that plumbagin suppressed migratory potential of CaSki cells which could be due to the reduced expression and activity of MMP-2 and upregulation of TIMP2. Interestingly, plumbagin also downregulated UHRF1 expression. Transient silencing of UHRF1 like plumbagin, induced G2/M phase arrest, enhanced apoptosis and suppressed metastasis of CaSki cells suggesting the role of UHRF1 in mediating anti-cancer activities of plumbagin. Plumbagin at IC

Topics: Apoptosis; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Female; Humans; Matrix Metalloproteinase 2; Naphthoquinones; Proto-Oncogene Proteins c-akt; Ubiquitin-Protein Ligases; Uterine Cervical Neoplasms

An RP-HPLC (Reverse Phase-High-performance liquid chromatography) method for the quantitative estimation and validation of the plumbagin in the methanolic fraction of. For achieving good separation, the RP-HPLC method was carried out with reverse phase C18 column, using methanol and water as mobile phase in the ratio of 65:35 (v/v), at the flow rate of 1 mL/min. The detection wavelength was set at 265 nm.. The developed method was accurate, specific, precise, and reproducible. This RP-HPLC may be useful for quantitative estimation of the chemical constituents present in the plant extract as well as the quality assessment of the herbal product.

Topics: Chromatography, High Pressure Liquid; Naphthoquinones; Plant Roots; Plumbaginaceae

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: Ataxia Telangiectasia Mutated Proteins; Carcinoma, Hepatocellular; Cell Cycle Proteins; Checkpoint Kinase 2; DNA Damage; Humans; Liver Neoplasms; M Cells; Naphthoquinones; Oxidative Stress; Phosphorylation; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53

A sustained increase in the mortality of pancreatic cancer (PC) and sudden metastasis-related mortality is a cause for concern. Aberrant expression of epidermal growth factor (EGF) receptor (EGFR) is noted in several cases of PC metastasis. The present study is aimed at analyzing the expression of EGFR in PC and its relevance to the progression of PC. Despite the number of studies that have shown the benefits of plumbagin on PC cells, its role on cancer stem cells remains largely unknown. To this end, the study used an EGF micro-environment to make cancer stem cells in vitro and ascertained the role of plumbagin in mitigating the actions of EGF. The kaplan-meier (KM) plot indicated reduced overall survival (OS) analysis in PC patients with high EGFR than low EGFR expression. Plumbagin pre-treatment significantly prevented EGF-induced survival, epithelial-to-mesenchymal transition (EMT), clonogenesis, migration, matrix metalloproteinase -2 (MMP-2) gene expression and its secretion, and matrix protein hyaluron production in PANC-1 cells. The computational studies indicate the greater affinity of plumbagin with different domains of EGFR than gefitinib. Several hallmarks of resistance and migration due to EGF are effectively attenuated by plumbagin. Collectively, these results warrant investigating the actions of plumbagin in a pre-clinical study to substantiate these findings.

Topics: Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Naphthoquinones; Pancreatic Neoplasms

Plumbagin is used in traditional medicine because of its anti-inflammatory and anti-microbial properties. As a naphthoquinone, plumbagin triggers the production of reactive oxygen species (ROS). In vitro cancer studies showed that plumbagin triggers apoptosis in cancer cells through ROS production. As cancer-mediated chronic inflammation can affect bone density, it was hypothesized that plumbagin might directly inhibit the formation of bone-resorbing osteoclasts. We previously showed that the effect of plumbagin on osteoclastogenesis differed between bone marrow-derived macrophages and the macrophage cell line RAW 264.7. Although RAW 264.7 macrophages are able to initiate the gene program required for osteoclastogenesis, only primary macrophages successfully differentiate into osteoclasts. Here, we show that RAW 264.7 cells are more sensitive toward plumbagin-induced apoptosis. In the presence of plumbagin and the cytokine RANKL, which triggers ROS production to drive osteoclastogenesis, RAW 264.7 macrophages produce increased amounts of ROS and die. Addition of the ROS scavenger N-acetyl cysteine prevented cell death, linking the failure to differentiate to increased ROS levels. RAW 264.7 cells show reduced expression of genes protective against oxidative stress, while primary macrophages have a higher tolerance toward ROS. Our data suggest that it is indispensable to consider cell (line)-intrinsic properties when studying phytochemicals.

Topics: Cell Differentiation; Naphthoquinones; Osteoclasts; RANK Ligand; Reactive Oxygen Species

Thioredoxin reductase 1 (TrxR1 or TXNRD1) is a major enzyme in cellular redox regulation and is considered as a drug target for cancer therapy. Previous studies have reported that plumbagin caused reactive oxygen species (ROS)-dependent apoptosis via inhibiting TrxR1 activity or being reduced by TrxR1, leading to selectively cancer cell death. However, the mechanism of TrxR1-mediated redox cycling of plumbagin is obscure and the evidence for plumbagin targeting TrxR1 is still lacking. Herein, we demonstrated that TrxR1 catalyzed plumbagin reduction in both selenocysteine (Sec)-dependent and independent manners, and its activity relied on the intact N-terminal motif of TrxR1, but a high-efficiency reduction was supported by the C-terminal thiols. During the redox cycling of plumbagin, excessive ROS production was observed coupled with oxygen. Using LC-MS and TrxR1 mutants, we found that the Sec residue of TrxR1 was modified by plumbagin, which converted the enzyme from antioxidant to pro-oxidant. Furthermore, we evaluated the therapeutic potential of plumbagin in non-small cell lung cancer (NSCLC), and found that Kelch-like ECH-associated protein 1 (KEAP1)-mutant NSCLC cells, which possess constitutive nuclear factor erythroid 2-related factor 2 (NRF2) activity, were insensitive to plumbagin; however, inhibition of glucose transporter 1 (GLUT1) by small-molecule BAY-876 or inhibiting glucose-6-phosphate dehydrogenase (G6PD) by 6-aminonicotinamide (6-AN) overcame the plumbagin-resistance of KEAP1-mutant NSCLC cells. Taken together, this study elucidated the pharmacological mechanism of plumbagin by targeting TrxR1 and revealed the synergy effect of plumbagin and BAY-876, which may be helpful for applying naphthoquinone compounds to chemotherapy, particularly for treating KEAP1-mutant NSCLC cells.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Glucose Transporter Type 1; Humans; Kelch-Like ECH-Associated Protein 1; Lung Neoplasms; Naphthoquinones; NF-E2-Related Factor 2; Pyrazoles; Quinolines; Rats; Reactive Oxygen Species; Selenocysteine; Thioredoxin Reductase 1

Plumbagin, a bioactive naphthoquinone, has demonstrated potent antitumor potential. However, plumbagin is a sparingly water-soluble compound; therefore, clinical translation requires and will be facilitated by the development of a new pharmaceutical formulation. We have generated an oil-in-water nanoemulsion formulation of plumbagin using a low-energy spontaneous emulsification process with propylene glycol caprylate (Capryol 90) as an oil phase and Labrasol/Kolliphor RH40 as surfactant and cosurfactant excipients. Formulation studies using Capryol 90/Labrasol/Kolliphor RH40 components, based on pseudoternary diagram and analysis of particle size distribution and polydispersity determined by dynamic light scattering (DLS), identified an optimized composition of excipients for nanoparticle formulation. The nanoemulsion loaded with plumbagin as an active pharmaceutical ingredient had an average hydrodynamic diameter of 30.9 nm with narrow polydispersity. The nanoemulsion exhibited long-term stability, as well as good retention of particle size in simulated physiological environments. Furthermore, plumbagin-loaded nanoemulsion showed an augmented cytotoxicity against prostate cancer cells PTEN-P2 in comparison to free drug. In conclusion, we generated a formulation of plumbagin with high loading drug capacity, robust stability, and scalable production. Novel Capryol 90-based nanoemulsion formulation of plumbagin demonstrated antiproliferative activity against prostate cancer cells, warranting thus further pharmaceutical development.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Carriers; Emulsions; Male; Mice; Nanoparticles; Naphthoquinones; Propylene Glycol; Prostatic Neoplasms

Osteoporosis is caused by enhanced bone resorption and relatively reduced bone formation. There is an unmet need to develop new agents with both antiresorptive and anabolic effects to treat osteoporosis, although drugs with either effect alone are available. A small molecular compound, plumbagin, was reported to inhibit receptor activator of nuclear factor kappa-B ligand-induced osteoclast (OC) differentiation by inhibiting IκBα phosphorylation-mediated canonical NF-κB activation. However, the key transcriptional factor RelA/p65 in canonical NF-κB pathway functions to promote OC precursor survival but not terminal OC differentiation. Here, we found that plumbagin inhibited the activity of NF-κB inducing kinase, the key molecule that controls noncanonical NF-κB signaling, in an ATP/ADP-based kinase assay. Consistent with this, plumbagin inhibited processing of NF-κB2 p100 to p52 in the progenitor cells of both OCs and osteoblasts (OBs). Interestingly, plumbagin not only inhibited OC but also stimulated OB differentiation in vitro. Importantly, plumbagin prevented trabecular bone loss in ovariectomized mice. This was associated with decreased OC surfaces on trabecular surface and increased parameters of OBs, including OB surface on trabecular surface, bone formation rate, and level of serum osteocalcin, compared to vehicle-treated mice. In summary, we conclude that plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects on bone and could represent a new class of agent for the prevention and treatment of osteoporosis.

Topics: Animals; Bone Density Conservation Agents; Female; Humans; Mice; Naphthoquinones; NF-kappa B; NF-kappaB-Inducing Kinase; Osteoclasts; Osteoporosis, Postmenopausal; Protein Serine-Threonine Kinases

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), can infect humans, terrestrial animals and fish. The emergence of bacterial resistance of S. agalactiae to antibiotics leads to an urgent need of exploration of new antimicrobial agents. In the study, the antibacterial activity of natural component plumbagin (PLB) against S. agalactiae was investigated in vitro and in vivo. The results showed that the minimal inhibitory concentration (MIC) of PLB against S. agalactiae was 8 mg/L. The growth curve assay revealed that PLB could inhibit the growth of S. agalactiae. In addition, the time-killing curve showed that S. agalactiae was killed almost completely by 2-fold MIC of PLB within 12 h. Transmission electron microscopy results showed obvious severe morphological destruction and abnormal cells of S. agalactiae after treated with PLB. The pathogenicity of S. agalactiae to zebrafish was significantly decreased after preincubation with PLB for 2 h in vitro, further indicating the bactericidal activity of PLB. Interestingly, PLB could kill S. agalactiae without inducing resistance development. Furthermore, pretreatment and post-treatment assays suggested that PLB also exhibited the antibacterial activity against S. agalactiae infection in vivo by effectively reducing the bacterial load and improving the survival rate of S. agalactiae-infected zebrafish. In summary, PLB had potent antibacterial activity against S. agalactiae in vitro and in vivo, and it could be an excellent antimicrobial candidate to prevent and control S. agalactiae infection.

Topics: Animals; Anti-Bacterial Agents; Fish Diseases; Naphthoquinones; Streptococcal Infections; Streptococcus agalactiae; Zebrafish

Plumbagin, a hydroxy-1,4-naphthoquinone, confers neuroprotection via antioxidant and anti-inflammatory properties. The present study aimed to assess the effect of plumbagin on behavioral and memory deficits induced by intrahippocampal administration of Quinolinic acid (QA) in male Wistar rats and reveal the associated mechanisms. QA (300 nM/4 μL in Normal saline) was administered i.c.v. in the hippocampus. QA administration caused depression-like behavior (forced swim test and tail suspension tests), anxiety-like behavior (open field test and elevated plus maze), and elevated anhedonia behavior (sucrose preference test). Furthermore, oxidative-nitrosative stress (increased nitrite content and lipid peroxidation with reduction of GSH), inflammation (increased IL-1β), cholinergic dysfunction, and mitochondrial complex (I, II, and IV) dysfunction were observed in the hippocampus region of QA-treated rats as compared to normal controls. Plumbagin (10 and 20 mg/kg; p.o.) treatment for 21 days significantly ameliorated behavioral and memory deficits in QA-administered rats. Moreover, plumbagin treatment restored the GSH level and reduced the MDA and nitrite level in the hippocampus. Furthermore, QA-induced cholinergic dysfunction and mitochondrial impairment were found to be ameliorated by plumbagin treatment. In conclusion, our results suggested that plumbagin offers a neuroprotective potential that could serve as a promising pharmacological approach to mitigate neurobehavioral changes associated with neurodegeneration.

Topics: Animals; Behavior, Animal; Depression; Male; Memory Disorders; Naphthoquinones; Oxidative Stress; Quinolinic Acid; Rats; Rats, Wistar

Nasopharyngeal carcinoma (NPC) is publicly known as a malignant tumor. Our previous study reported that plumbagin exhibits potent anti-cancer actions. Nevertheless, more mechanical details of plumbagin against NPC remain unknown. The present study aimed to unmask the core targets/genes and anti-NPC mechanisms involved in the signaling pathways of plumbagin prior to biochemical validation.. A network pharmacology approach was employed to respective identification of mutual and core targets/genes in plumbagin and/treating NPC. Molecular docking determination was used to identify core target proteins for biochemical validation using human and cell line samples.. In total, 60 anti-NPC genes of plumbagin were screened out, and then nine core target genes of plumbagin against NPC were identified accordingly. The enrichment findings revealed detailed biological functions and pharmacological pathways of plumbagin against NPC. Moreover, in silico analysis using molecular docking had determined the core targets for further experimental validation, comprising protein kinase B (AKT1) and sarcoma gene (SRC). In human sample validation, clinical NPC sections showed increased positive expression of AKT1 and SRC. Additionally, plumbagin-treated NPC cells resulted in inactivated protein expression of AKT1 and SRC.. The re-identified core targets/genes in the molecular docking report may function as plumbagin-related pharmacological targets for treating NPC via experimental validation. Furthermore, additional anti-NPC molecular mechanisms of plumbagin action were disclosed on the basis of enrichment findings. © 2022 Society of Chemical Industry.

Topics: Humans; Molecular Docking Simulation; Naphthoquinones; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms

Ferroptosis has attracted increasing interest in cancer therapy. Emerging evidences suggest that naturally occurring naphthoquinones exhibit potent anti-glioma effects via various mechanisms.. The anti-glioma effects of plumbagin were evaluated by in vitro and in vivo experiments. Anti-glioma mechanism of plumbagin was studied by proteomics, flow cytometry, MDA assay, western blot, and RT-PCR. Gene knockdown/overexpression, molecular docking, PharmMappper database, and coimmunoprecipitation were used to study the targets of plumbagin.. Plumbagin showed higher blood-brain barrier penetration ability than that of lapachol and shikonin and elicited significant growth inhibitory effects in vitro and in vivo. Ferroptosis was the main mechanism of plumbagin-induced cell death. Mechanistically, plumbagin significantly downregulated the protein and mRNA levels of xCT and decreased GPX4 protein levels. NAD(P)H quinone dehydrogenase 1 (NQO1) was revealed as a plumbagin predictive target using PharmMappper database and molecular docking. Plumbagin enhanced NQO1 activity and decreased xCT expression, resulting in NQO1-dependent cell death. It also induced GPX4 degradation via the lysosome pathway and caused GPX4-dependent cell death.. Plumbagin inhibited in vitro and in vivo glioma growth via targeting NQO1/GPX4-mediated ferroptosis, which might be developed as a novel ferroptosis inducer or anti-glioma candidate.

Topics: Cell Line, Tumor; Ferroptosis; Glioma; Humans; Molecular Docking Simulation; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Phospholipid Hydroperoxide Glutathione Peroxidase

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Cycle Proteins; Coordination Complexes; Energy Metabolism; Humans; Ligands; Mice; Mice, Nude; Naphthoquinones; Ruthenium

Plumbagin, a natural bicyclic naphthoquinone, has diverse pharmacological properties and biological benefits against a number of disorders, including liver disease. Though plumbagin's hepatoprotective potential attracts attention, currently no experimental evidence exists on its effectiveness against cholestatic liver injury. The present study investigated its hepatoprotection in the rat model of extrahepatic cholestasis using Bile Duct Ligation (BDL). We found that daily plumbagin supplementation protected the liver from cholestatic damage. Hepatoprotective actions of plumbagin were accompanied by reduction of Transforming Growth Factor β1 (TGF-β1)/Smad, High Mobility Group Box-1 (HMGB1)/Toll-Like Receptor-4 (TLR4), Hypoxia-Inducible Factor-1α (HIF-1α), Aryl Hydrocarbon Receptor (AhR), Heat Shock Protein 90 (HSP90), caveolin-1, NF-κB/AP-1, Dynamin Related Protein-1 (Drp1), malondialdehyde level, Interleukin-1β (IL-1β), p62/SQSTM1, and caspase 3 as well as increase of Farnesoid X Receptor (FXR), bile acid efflux transporters, glutathione, LC3-II, Beclin1, and nuclear NF-E2-Related Factor-2 (Nrf2) and Transcription Factor EB (TFEB). The activation of nuclear Nrf2 caused by plumbagin correlated well with the improvement in bile acid retention, liver histology, serum biochemical, ductular reaction, mitochondrial dysfunction, oxidative stress, inflammation, apoptosis, impaired autophagy, and fibrosis, involving interplay of multiple intracellular signaling pathways. Plumbagin is likely a candidate drug to protect the liver from cholestatic damages. Despite the promising findings from this study, translational implication of plumbagin on cholestatic liver injury warrants further investigation.

Topics: Animals; Bile Acids and Salts; Bile Ducts; Cholestasis; Disease Models, Animal; Ligation; Liver; Naphthoquinones; NF-E2-Related Factor 2; Rats

This study aimed to explore the effects of plumbagin on rheumatoid arthritis (RA) and its mechanism. The RA cell model was simulated following the treatment of interleukin-1β (IL-1β). After the treatment of various concentrations of plumbagin, the impact of plumbagin on the cell viability was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The collagen-induced arthritis (CIA) model was established using the solution of bovine type II collagen. Hematoxylin-eosin staining was used to observe the changes of ankle joint tissue, while enzyme-linked immunosorbent assay and western blot were applied to detect the level of inflammatory cytokines. Plumbagin inhibited the viability of human fibroblast-like synoviocytes (HFLS) at the concentration of 1 ~ 3.5 μM. The inhibitory effect of 1 μM plumbagin on cell proliferation was similar to that of methotrexate, the drug used as the positive control. Plumbagin downregulated the levels of inflammatory cytokines and matrix metalloproteinases (MMPs) in IL-1β-treated HFLS, and suppressed the activation of IκB and nuclear factor kappa-B (NF-κB) as well as the entry of p65 into the nucleus. It was also demonstrated in animal experiments that plumbagin inhibited the activation of NF-κB pathway, down-regulated the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and MMPs, and alleviated joint damage in CIA-modeled mice. Collectively speaking, plumbagin might down-regulate the levels of inflammatory cytokines and MMPs through inhibiting the activation of the NF-κB pathway, thereby attenuating RA-induced damage to cells and joints.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cattle; Cells, Cultured; Cytokines; Interleukin-1beta; Interleukin-6; Matrix Metalloproteinases; Mice; Naphthoquinones; NF-kappa B; Synovial Membrane; Tumor Necrosis Factor-alpha

Hepatocellular carcinoma (HCC) is resistant to current immunotherapy. This poor outcome mainly results from the immunosuppressive characteristics of tumor microenvironment (TME). Accumulating evidence indicates that some chemotherapy agents trigger immunogenic cell death (ICD), providing a promising strategy to remodel the immunosuppressive TME. The role of Plumbagin (PLB, a naphthoquinone compound from Plumbago zeylanica L.) as the ICD inducer for HCC cells was confirmed in this study. Dihydrotanshinone I (DIH, a phenanthraquinone compound of Salvia miltiorrhiza) functioned as the ICD enhancer by generating the reactive oxygen species (ROS). A poly(D,L-lactic-co-glycolic acid) (PLGA)-based nanoparticle (NP) was used to co-encapsulate PLB, DIH and NH

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Furans; Lactic Acid; Liver Neoplasms; Mice; Nanoparticles; Naphthoquinones; Phenanthrenes; Quinones; Tumor Microenvironment

Topics: Antifungal Agents; Biofilms; Cryptococcosis; Cryptococcus neoformans; Microbial Sensitivity Tests; Naphthoquinones

In recent years, derivatives of natural compounds are synthesized to increase the bioavailability, pharmacology, and pharmacokinetics properties. The naphthoquinone, plumbagin (PLU), is well known for its anticancer activity. However, the clinical use of PLU is hindered due to its toxicity. Previous reports have shown that modification of PLU at 5'-hydroxyl group has reduced its toxicity towards normal cell line. In accordance, in the present study, 5'-hydroxyl group of PLU was esterified with S-allyl cysteine (SAC) to obtain PLU-SAC ester. The drug-likeness of PLU-SAC was understood by in silico ADME analysis. PLU-SAC was characterized by UV-visible spectroscopy, mass spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Molecular docking and dynamics simulation analysis revealed the interaction of PLU-SAC with proteins of interest in cancer therapy such as human estrogen receptor α, tumor protein p53 negative regulator mouse double minute 2, and cyclin-dependent kinase 2. MMGBSA calculation showed the favorable binding energy which in turn demonstrated the stable binding of PLU-SAC with these proteins. PLU-SAC showed apoptosis in breast cancer cell line (MCF-7) by inducing oxidative stress, disturbing mitochondrial function, arresting cells at G1 phase of cell cycle, and initiating DNA fragmentation. However, PLU-SAC did not show toxicity towards normal Vero cell line. PLU-SAC was synthesized and structurally characterized, and its anticancer activity was determined by in silico and in vitro analysis.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cysteine; Esters; Humans; Mice; Molecular Docking Simulation; Naphthoquinones

Plumbagozeylanica grows widely in many tropical countries. In Indonesia, this plant, known as Daun Encok, has some beneficial effects on human health.

Topics: Humans; Indonesia; Naphthoquinones; Phytochemicals; Plant Extracts; Plumbaginaceae

Snails of the genus Biomphalaria are intermediate hosts of Schistosoma mansoni, the main etiological agent of schistosomiasis mansoni, which affects about 236.6 million people in tropical and subtropical regions of the world. The World Health Organization recommends the population control of vector snails as one of the strategies to reduce the prevalence and incidence of schistosomiasis. In this study, molluscicidal and antiparasitic activities of plumbagin, a naturally sourced naphthoquinone with a range of biological effects, were evaluated against B. glabrata and cercariae of S. mansoni.. After 24 h of exposure, plumbagin demonstrated molluscicidal activity at low concentrations against embryos (LC. Plumbagin proved to be a promising substance for the control of B. glabrata population, intermediate host of S. mansoni, as well as the cercariae, infective stage for humans (definitive host), while being moderately toxic to A. salina, a crustacean widely used in ecotoxicity tests. © 2022 Society of Chemical Industry.

Topics: Animals; Biomphalaria; DNA Damage; Humans; Naphthoquinones; Schistosomiasis mansoni

Schistosomiasis is still a major health problem affecting nearly 250 million people worldwide and causes approximately 280,000 deaths per year. The disease causes a serious granulomatous inflammatory response that produces significant mortality. Plumbagin reportedly displays anti-inflammatory, anti-fibrotic, antioxidant and anthelmintic properties. This study further elucidates these properties. Mice were infected with schistosomes and divided into five groups: non-infected untreated (C); infected untreated (IU); non-infected treated with plumbagin (P); infected treated with plumbagin (PI) and infected treated with praziquantel (PZ). Mice treated with 20 mg plumbagin/kg body weight showed reduction of 64.28% and 59.88% in male and female animals, respectively. Also, the number of eggs/g tissue was reduced 69.39%, 68.79% and 69.11% in liver, intestine and liver/intestine combined, respectively. Plumbagin alleviated schistosome-induced hepatosplenomegaly and reduced hepatic granuloma and liver collagen content by 62.5% and 35.26%, respectively while PZQ reduced hepatic granuloma and liver collagen content by 41.11% and 11.21%, respectively. Further, plumbagin treatment significantly (p < .001) reduced IL-4, IL-13, IL-17, IL-37, IFN-γ, TGF-β and TNF-α levels and significantly (p < .001) upregulated IL-10. Plumbagin treatment restored hepatic enzymes activity to nearly normal levels and induced an increase in catalase, SOD, GSH, total thiol and GST in liver tissue homogenate. NO and LPO content was, however, decreased. Moreover, serum IgG levels significantly increased. The present study is the first to report immunomodulatory and schistosomicidal activities of plumbagin in schistosomiasis.

Topics: Animals; Anthelmintics; Anti-Inflammatory Agents; Antioxidants; Catalase; Female; Granuloma; Immunoglobulin G; Interleukin-10; Interleukin-13; Interleukin-17; Interleukin-4; Liver; Male; Mice; Naphthoquinones; Praziquantel; Schistosoma mansoni; Schistosomiasis; Schistosomiasis mansoni; Schistosomicides; Sulfhydryl Compounds; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Female; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

A sensitive and rapid UPLC-MS/MS method for determination of tazemetostat in rat plasma was developed, and the pharmacokinetics of herb-drug interactions (HDIs) of plumbagin (PLB) and tazemetostat was investigated.. In the calibrated range of 1-1000 ng/mL, tazemetostat had a good linearity. The extraction recovery was more than 84%, and the RSD of intra-batch and inter-batch precision were both less than 15%. The C. A simple, rapid and sensitive UPLC-MS/MS method for the determination of tazemetostat in rat plasma was established. PLB can inhibit the metabolism of tazemetostat and increase the plasma exposure of tazemetostat in rats.

Topics: Acetonitriles; Animals; Benzamides; Biphenyl Compounds; Carboxymethylcellulose Sodium; Chromatography, High Pressure Liquid; Chromatography, Liquid; Herb-Drug Interactions; Morpholines; Naphthoquinones; Pyridones; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sodium; Tandem Mass Spectrometry

Novel cytochrome P450s, CYP81B140 and CYP81B141 from Plumbago zeylanica were functionally characterized to understand their involvement in polyketide plumbagin biosynthesis. Further, we propose 3-methyl-1-8-naphthalenediol and isoshinanolone as intermediates for plumbagin biosynthesis. Plumbago zeylanica L. (P. zeylanica) is a medicinally important plant belonging to the family Plumbaginaceae. It comprises the most abundant naphthoquinone plumbagin having anti-cancer activity. Only the polyketide synthase (PKS) enzyme has been identified from the biosynthetic pathway which catalyzes iterative condensation of acetyl-CoA and malonyl-CoA molecules. The plumbagin biosynthesis involves hydroxylation, oxidation, hydration and dehydration of intermediate compounds which are expected to be catalyzed by cytochrome P450s (CYPs). To identify the CYPs, co-expression analysis was carried out using PKS as a candidate gene. Out of the eight identified CYPs, CYP81B140 and CYP81B141 have similar expression with PKS and belong to the CYP81 family. Phylogenetic analysis suggested that CYP81B140 and CYP81B141 cluster with CYPs from CYP81B, CYP81D, CYP81E and CYP81AA subfamilies which are known to be involved in the hydroxylation and oxidation reactions. Moreover, artificial microRNA-mediated transient individual silencing and co-silencing of CYP81B140 and CYP81B141 significantly reduced plumbagin and increased the 3-methyl-1-8-naphthalenediol and isoshinanolone content. Based on metabolite analysis, we proposed that 3-methyl-1-8-naphthalenediol and isoshinanolone function as intermediates for plumbagin biosynthesis. Transient silencing, over-expression and docking analysis revealed that CYP81B140 is involved in C-1 oxidation, C-4 hydroxylation and [C2-C3] hydration of 3-methyl-1-8-naphthalenediol to form isoshinanolone, whereas CYP81B141 is catalyzing [C2-C3] dehydration and C-4 oxidation of isoshinanolone to form plumbagin. Our results indicated that both CYP81B140 and CYP81B141 are promiscuous and necessary for plumbagin biosynthesis. This is the first report of identification and functional characterization of P. zeylanica-specific CYPs involved in plumbagin biosynthetic pathway and in general hexaketide synthesis in plants.

Topics: Acetyl Coenzyme A; Cytochromes; Dehydration; Genomics; MicroRNAs; Naphthoquinones; Phylogeny; Plant Roots; Plumbaginaceae; Polyketide Synthases; Polyketides

The survival of ovary granulosa cells (GC) is critical in the initiation and progression of polycystic ovary syndrome (PCOS) in females. Here, we found that the PCOS process is accompanied by massive GC pyroptosis resulting from Caspase-1 inflammasome activation. Administration of plumbagin, an effective compound isolated from plant medicine, can prevent the pyroptosis of GC and the onset of PCOS. Mechanistic study indicates the over-activation of the inflammasome in GC is due to the upregulation of WTAP, a key regulator of the RNA N6-methylase complex. WTAP mediates the mRNA N6-methylation of NLRP3 inflammasome component ASC and enhances ASC RNA stability, which results in the overactivation of the inflammasome in GCs from the PCOS model. Plumbagin treatment suppresses the WTAP-mediated N6-methylation of ASC mRNA and reduces the pyroptosis of GCs. This study supports the profound potential of plumbagin in PCOS treatment.

Topics: Cell Cycle Proteins; Female; Granulosa Cells; Humans; Inflammasomes; Methylation; Naphthoquinones; Polycystic Ovary Syndrome; Pyroptosis; RNA Splicing Factors; RNA, Messenger

The most abundant active compound in Droseraceae is plumbagin, a naphthoquinone widely used for medical purposes due to its antimicrobial, antitussive, antimalarial and anticancer properties. In this work, we created a light-emitting diode (LED) based culture illumination setup as an alternative to fluorescent lamps traditionally used as a light source in plant in vitro cultures. The plants of Drosera binata and Drosera peltata cultured under LED illumination grew equally well and produced similar amounts of biologically active compounds as plants grown under fluorescent lamps. The plants were cultured on two media differing in mineral composition, sucrose content and pH. Secondary metabolites were extracted with ethanol from the plants after harvesting. The extracts were subjected to HPLC and microbiological analyses. We observed differences in morphology and secondary metabolism between plants of the same species grown on different media. However, we did not note significant changes in secondary metabolite yield under assessed lighting conditions. We propose LEDs as a more efficient, eco-friendly and economically reasonable source of light for big scale in vitro production of plumbagin in Drosera species than fluorescent lamps.

Topics: Anti-Infective Agents; Candida albicans; Culture Media; Droseraceae; Light; Lighting; Microbial Sensitivity Tests; Naphthoquinones; Salmonella enterica; Staphylococcus aureus

We investigated the cytotoxic effects of plumbagin on metastatic retinoblastoma, using the highly metastatic cell line Y79.. Effect of plumbagin on cell growth was assessed with water-soluble tetrazolium 1 (WST-1) cell proliferation assay and automated hemocytometry with trypan blue-exclusion assay. Cell death was studied with acridine orange/ethidium bromide live-dead assay and annexin-V-fluorescein isothiocyanate/propidium iodide microscopy. Loss of mitochondrial membrane potential was studied with JC-10 dye and caspase activation was investigated using CellEvent Caspase-3/7 Green detection reagent.. Plumbagin highly significantly reduced the growth of Y79 cells treated for 24 h with 2.5 μM or more. Plumbagin also induced significantly high levels of cell death which was associated with loss of mitochondrial membrane potential and caspase activation.. At very low concentration (2.5 μM), plumbagin potently induced cytotoxicity in metastatic retinoblastoma cells via loss of mitochondrial membrane potential and caspase activation.

Topics: Antineoplastic Agents, Phytogenic; Caspases; Cell Death; Cell Line, Tumor; Cell Proliferation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Neoplasm Metastasis; Retinal Neoplasms; Retinoblastoma

MSMEG_2295 is a TetR family protein encoded by the first gene of a

Topics: Bacterial Proteins; DNA, Bacterial; Gene Expression Regulation, Bacterial; Mutation; Mycobacterium smegmatis; Naphthoquinones; Operator Regions, Genetic; Operon; Promoter Regions, Genetic; Pyruvic Acid; Repressor Proteins; Superoxides

Carnivorous plants feed on animal prey, mainly insects, to get additional nutrients. This carnivorous syndrome is widely investigated and reported. In contrast, reports on herbivores feeding on carnivorous plants and related defenses of the plants under attack are rare. Here, we studied the interaction of a pitcher plant, Nepenthes x ventrata, with a generalist lepidopteran herbivore, Spodoptera littoralis, using a combination of LC/MS-based chemical analytics, choice and feeding assays. Chemical defenses in N. x ventrata leaves were analyzed upon S. littoralis feeding. A naphthoquinone, plumbagin, was identified in Nepenthes defense against herbivores and as the compound mainly responsible for the finding that S. littoralis larvae gained almost no weight when feeding on Nepenthes leaves. Plumbagin is constitutively present but further 3-fold increased upon long-term (> 1 day) feeding. Moreover, in parallel de novo induced trypsin protease inhibitor (TI) activity was identified. In contrast to TI activity, enhanced plumbagin levels were not phytohormone inducible, not even by defense-related jasmonates although upon herbivory their level increased more than 50-fold in the case of the bioactive jasmonic acid-isoleucine. We conclude that Nepenthes is efficiently protected against insect herbivores by naphthoquinones acting as phytoanticipins, which is supported by additional inducible defenses. The regulation of these defenses remains to be investigated.

Topics: Abscisic Acid; Animals; Carnivorous Plant; Cyclopentanes; Diet; Herbivory; Larva; Naphthoquinones; Oxylipins; Phytochemicals; Plant Growth Regulators; Plant Leaves; Protease Inhibitors; Salicylic Acid; Sarraceniaceae; Spodoptera

Clinically, evidence shows that uterine corpus endometrial carcinoma (UCEC) patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may have a higher death-rate. However, current anti-UCEC/coronavirus disease 2019 (COVID-19) treatment is lacking. Plumbagin (PLB), a pharmacologically active alkaloid, is an emerging anti-cancer inhibitor. Accordingly, the current report was designed to identify and characterize the anti-UCEC function and mechanism of PLB in the treatment of patients infected with SARS-CoV-2

Topics: Adult; Aged; Aged, 80 and over; Calcium-Binding Proteins; Carcinoma, Endometrioid; Computational Biology; COVID-19; COVID-19 Drug Treatment; Drug Screening Assays, Antitumor; Endometrial Neoplasms; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genetic Association Studies; Host-Pathogen Interactions; Humans; Membrane Proteins; Middle Aged; Mitogen-Activated Protein Kinase 3; Molecular Docking Simulation; Naphthoquinones; Prognosis; SARS-CoV-2; Signal Transduction; Tumor Necrosis Factor-alpha; Uterus

Plumbago europaea L. is a plant utilized in Palestinian ethnomedicine for the treatment of various dermatological diseases. The current investigation was designed to isolate plumbagin from P. europaea leaves, roots and for the first time from the stems. Moreover, it aimed to evaluate the antimycotic activity against three human fungal pathogens causing dermatophytosis, also against an animal fungal pathogen. The qualitative analysis of plumbagin from the leaves, stems, and roots was conducted using HPLC and spectrophotometer techniques, while the structure of plumbagin was established utilizing Proton and Carbon-13 Nuclear Magnetic Resonance (NMR) and Infrared (IR) techniques. The entire plant constituents were determined by GC-MS. Moreover, the antimycotic activity against Ascosphaera apis, Microsporum canis, Trichophyton rubrum, and Trichophyton mentagrophytes was assessed utilizing the poison food technique method. The percentage of plumbagin recorded in the leaves, stems, and roots was found to be 0.51±0.001%, 0.16±0.001%, and 1.65±0.015%, respectively. The GC-MS examination declared the presence of 59 molecules in the plant extract. The plant extract and pure plumbagin exhibited complete inhibition against all tested dermatophytes at 6.0mg/mL for the extracts and 0.2mg/mL for plumbagin. P. europaea root is the best source of plumbagin and the plant extract could represent a potential drug candidate for the treatment of dermatophytosis infections. Further studies required to design suitable dosage forms from the natural P. europaea root extracts or plumbagin alone, to be utilized for the treatment of dermatological and veterinary ailments.

Topics: Antifungal Agents; Arthrodermataceae; Chromatography, High Pressure Liquid; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Microsporum; Molecular Structure; Naphthoquinones; Onygenales; Plant Leaves; Plant Roots; Plant Stems; Plumbaginaceae; Spectrophotometry, Infrared

Stable encapsulation of medically active compounds can lead to longer storage life and facilitate the slow-release mechanism. In this work, the dynamic and molecular interactions between plumbagin molecule with β-cyclodextrin (BCD) and its two derivatives, which are dimethyl-β-cyclodextrin (MBCD), and 2-

Topics: 2-Hydroxypropyl-beta-cyclodextrin; beta-Cyclodextrins; Computational Biology; Cyclodextrins; Drug Compounding; Molecular Dynamics Simulation; Naphthoquinones; Solubility; Water

<b>Background and Objective:</b> The medicinal herb <i>Plumbago indica</i> (PI) and its major constituent plumbagin have reported pharmacological properties but there is a lack of information about their herb-drug interactions. The effects of methanolic (PI-MeOH) and ethanolic (PI-EtOH) crude extracts of PI and plumbagin on the expression of cytochrome P450s (<i>CYP1A2</i>, <i>CYP2E1</i> and <i>CYP3A4</i>) and transporters (<i>ABCC1</i>, <i>ABCG2</i> and <i>SLC22A11</i>) were investigated in BeWo and HepG2 cells. <b>Materials and Methods:</b> BeWo or HepG2 cells were treated with 0.5-5 μM plumbagin or 25-500 μg mL¹ of PI-MeOH or PI-EtOH for 24 hrs. Total RNA was extracted and mRNA expression of CYPs and transporters were determined using RT-qPCR. <b>Results:</b> PI and plumbagin affected mRNA expression differently in the two tested cell types. In BeWo cells, all concentrations of PI-MeOH induced <i>CYP2E1</i>, 100 and 500 μg Ml¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i>, <i>CYP3A4 </i>and <i>ABCG2 </i>and 500 μg mL¹ PI-EtOH induced <i>ABCG2</i> expression. Plumbagin suppressed <i>CYP1A2</i> and induced <i>SLC22A11 </i>expression at the highest concentration, 5 μM. In HepG2 cells, 5 μM plumbagin and 500 μg Ml¹ PI-EtOH suppressed <i>CYP3A4 </i>expression and 500 μg mL¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i> and <i>CYP2E1 </i>expression. <i>ABCC1</i> expression was induced by all treatments while <i>ABCG2</i> and <i>SLC22A11 </i>were induced only by 500 μg mL¹ PI-MeOH and PI-EtOH. <b>Conclusion:</b> The use of PI or plumbagin supplements in large quantities or for long periods should be carefully considered due to the risk of herbal drug interactions via modulated expression of CYPs and transporters.

Topics: Cytochrome P-450 Enzyme System; Hep G2 Cells; Humans; Naphthoquinones; Plumbaginaceae

In this perspective, we propose to leverage reactive oxygen species (ROS) induction as a potential therapeutic measure against viral infections. Our rationale for targeting RNA viral infections by pro-oxidants is routed on the mechanistic hypothesis that ROS based treatment paradigm could impair RNA integrity faster than the other macromolecules. Though antiviral drugs with antioxidant properties confer potential abilities for preventing viral entry, those with pro-oxidant properties could induce the degradation of nascent viral RNA within the host cells, as RNAs are highly prone to ROS mediated degradation than DNA/proteins. We have previously established that Plumbagin is a highly potent ROS inducer, which acts through shifting of the host redox potential. Besides, it has been reported that Plumbagin treatment has the potential for interrupting viral RNA replication within the host cells. Since the on-going Corona Virus Disease - 2019 (COVID-19) global pandemic mediated by Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) exhibits high infectivity, the development of appropriate antiviral therapeutic strategies remains to be an urgent unmet race against time. Therefore, additional experimental validation is warranted to determine the appropriateness of repurposable drug candidates, possibly ROS inducers, for fighting the pandemic which could lead to saving many lives from being lost to COVID-19.

Topics: Animals; Antiviral Agents; COVID-19; COVID-19 Drug Treatment; Humans; Naphthoquinones; Pandemics; Reactive Oxygen Species; RNA, Viral; SARS-CoV-2; Virus Diseases

Plumbagin (PLB) is a naphthoquinone endowed with potential medicinal properties, including anticancer activities. We evaluated the effects of PLB on the viability, cell cycle, autophagy, and apoptosis of endometrial carcinoma Ishikawa cells. The proliferation of cells was significantly inhibited by PLB at 0, 8, 10, and 12 μM. By up regulating the expression of p53 and p21, PLB could block the cell cycle in G2/M phase and down regulate cyclin dependent kinase. The apoptosis in the cancer cells was characterized by noticeable chromatin edge collection, nuclear membrane expansion, and vacuolization. PLB could significantly induce autophagy in cells, and its inhibition ability and apoptosis induction were weakened by the autophagy inhibitor SBI-0206965. Our study suggested that PLB may exert anticancer effects by abrogating PI3K/Akt pathway, which recommends it as a promising future phytotherapeutic candidate for EC treatment.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Cell Survival; Endometrial Neoplasms; Female; G2 Phase Cell Cycle Checkpoints; Humans; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Some plant species are less susceptible to herbivore infestation than others. The reason for this is often unknown in detail but is very likely due to an efficient composition of secondary plant metabolites. Strikingly, carnivorous plants of the genus

Topics: Animals; Caryophyllales; Insecticides; Larva; Naphthoquinones; Plant Leaves

To evaluate the inhibitory effect and mechanism of plumbagin (PLB) against drug-resistant tongue squamous cell carcinoma (TSCC), and whether its antitumour effect is not affected by tumour drug resistance.. TSCC sensitive CAL27 cells and drug-resistant CAL27/RE cells were used to study the cytotoxicity and mechanism of PLB in vitro, including CCK-8 analysis, colony formation, DAPI staining, flow cytometry assay, transmission electron microscopy, western blotting assay, autophagy, apoptosis and ROS fluorescent probes. BALB/c nude mice xenograft models were used to study the growth inhibitory effect of PLB in vivo.. The results showed that the cell viability and proliferation inhibition and apoptosis induction abilities of PLB on drug-resistant cells were more obvious than that on sensitive cells. And PLB induced protective autophagy in TSCC cells. Mechanistically, PLB induced apoptosis and autophagy by generating reactive oxygen species to mediate JNK and AKT/mTOR pathways. Finally, the growth inhibitory effect of PLB against drug-resistant TSCC was also confirmed in vivo.. PLB will be a promising anticancer agent to overcome drug-resistant TSCC without being affected by its drug resistance properties.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Male; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Phytotherapy; Plant Extracts; Tongue; Tongue Neoplasms; TOR Serine-Threonine Kinases

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it was reported that plumbagin may inhibit the differentiation of bone resorbing osteoclasts in cancer-related models, we wanted to elucidate whether plumbagin interferes with cytokine-induced osteoclastogenesis. Using C57BL/6 mice, we unexpectedly found that plumbagin treatment enhanced osteoclast formation and that this effect was most pronounced when cells were pre-treated for 24 h with plumbagin before subsequent M-CSF/RANKL stimulation. Plumbagin caused a fast induction of NFATc1 signalling and mTOR-dependent activation of p70S6 kinase which resulted in the initiation of protein translation. In line with this finding, we observed an increase in RANK surface expression after Plumbagin stimulation that enhanced the responsiveness for subsequent RANKL treatment. However, in Balb/c mice and Balb/c-derived RAW264.7 macrophages, these findings could not be corroborated and osteoclastogenesis was inhibited. Our results suggest that the effects of plumbagin depend on the model system used and can therefore either trigger or inhibit osteoclast formation.

Topics: Animals; Bone Resorption; Macrophage Colony-Stimulating Factor; Mice; Naphthoquinones; NFATC Transcription Factors; Osteoclasts; RANK Ligand; RAW 264.7 Cells; TOR Serine-Threonine Kinases

Although the biomedical sciences have achieved tremendous success in developing novel approaches to managing prostate cancer, this disease remains one of the major health concerns among men worldwide. Liposomal formulations of single drugs have shown promising results in cancer treatment; however, the use of multi drugs has shown a better therapeutic index than individual drugs. The identification of cancer-specific receptors has added value to design targeted drug delivering nanocarriers. We have developed genistein and plumbagin co-encapsulating liposomes (∼120 nm) with PSMA specific antibodies to target prostate cancer cells selectively in this work. These liposomes showed >90 % decrease in PSMA expressing prostate cancer cell proliferation without any appreciable toxicity to healthy cells and human red blood cells. Release of plumbagin and genistein was found to decrease the expression of PI3/AKT3 signaling proteins and Glut-1 receptors (inhibited glucose uptake and metabolism), respectively. The decrease in migration potential of cells and induced apoptosis established the observed anti-proliferative effect in prostate cancer cell lines. The discussed strategy of developing novel, non-toxic, and PSMA specific antibody conjugated liposomes carrying genistein and plumbagin drugs may also be used for encapsulating other drugs and inhibit the growth of different types of cancers.

Topics: Apoptosis; Cell Line, Tumor; Genistein; Glutamate Carboxypeptidase II; Humans; Liposomes; Male; Naphthoquinones; Prostatic Neoplasms

Plumbagin, a naphthoquinone extracted from the officinal leadwort presenting promising anti-cancer properties, has its therapeutic potential limited by its inability to reach tumors in a specific way at a therapeutic concentration following systemic injection. The purpose of this study is to assess whether a novel tumor-targeted, lipid-polymer hybrid nanoparticle formulation of plumbagin would suppress the growth of B16-F10 melanoma in vitro and in vivo.. Novel lipid-polymer hybrid nanoparticles entrapping plumbagin and conjugated with transferrin, whose receptors are present in abundance on many cancer cells, have been developed. Their cellular uptake, anti-proliferative and apoptosis efficacy were assessed on various cancer cell lines in vitro. Their therapeutic efficacy was evaluated in vivo after tail vein injection to mice bearing B16-F10 melanoma tumors.. The transferrin-bearing lipid-polymer hybrid nanoparticles loaded with plumbagin resulted in the disappearance of 40% of B16-F10 tumors and regression of 10% of the tumors following intravenous administration. They were well tolerated by the mice.. These therapeutic effects, therefore, make transferrin-bearing lipid-polymer hybrid nanoparticles entrapping plumbagin a highly promising anti-cancer nanomedicine.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Coumarins; Drug Liberation; Endocytosis; Female; Humans; Injections, Intravenous; Lipids; Melanoma, Experimental; Mice, Inbred BALB C; Nanoparticles; Naphthoquinones; Polymers; Skin Neoplasms; Thiazoles; Transferrin

The protein chromosome region maintenance 1 (CRM1) is an important nuclear export factor and drug target in diseases such as cancer and viral infections. Several plant-derived CRM1 inhibitors including plumbagin and oridonin possess potent antitumor activities. However, their modes of CRM1 inhibition remain unclear. Here, a multimutant CRM1 was engineered to enable crystallization of these two small molecules in its NES groove. Plumbagin and oridonin share the same three conjugation sites in CRM1. In solution, these two inhibitors targeted more CRM1 sites and inhibited its activity through promoting its aggregation, in addition to directly targeting the NES groove. While the plumbagin-bound NES groove resembles the NES-bound groove state, the oridonin complex reveals for the first time a more open NES groove. The observed greater NES groove dynamics may improve cargo loading through a "capture-and-tighten" mechanism. This work thus provides new insights on the mechanism of CRM1 inhibition by two natural products and a structural basis for further development of these or other CRM1 inhibitors.

Topics: Amino Acid Sequence; Diterpenes, Kaurane; Exportin 1 Protein; Karyopherins; Molecular Structure; Mutant Proteins; Naphthoquinones; Protein Engineering; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear

Osteoporosis is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the suppressive role of interleukin (IL)-35 in nuclear factor kappa-B ligand receptor activator (RANKL) and macrophage colony stimulating factor (M-CSF)-induced osteoclastogenesis and angiogenesis in osteoclasts.. Osteoclasts differentiation were induced by incubation of mouse leukemic monocyte/macrophage cell line RAW264.7 cells in the presence of RANKL and M-CSF and was assessed with tartrate-resistant acid phosphatase (TRAP) staining assay. The viability and apoptosis of RAW264.7 was measured using CCK-8 assay and flow cytometry, respectively. The expression of angiogenic genes and proteins were measured using RT-PCR, Western blots and ELISA. The inhibition of Th17/IL-17 axis was examined using plumbagin, which was demonstrated as an IL-17A related signaling pathway inhibitor.. IL-35 inhibited the viability of RAW264.7 cells and promoted the apoptosis of RAW264.7 cells in a dose-dependent manner. Furthermore, IL-35 dose-dependently suppressed the expression of angiogenic markers including VEGF and its receptor. The suppressive effect of IL-35 was confirmed through the activation of Th17/IL-17 axis.. We demonstrated for the first time the immuno-suppressive function of IL-35 on RANKL and M-CSF-induced osteoclastogenesis and angiogenesis through Th17/IL-17 axis. Therapeutic approach involving augmentation of IL-35 regulatory response may serve as a novel treatment option for osteoporosis, especially by suppressing bone resorption and angiogenesis.

Topics: Animals; Apoptosis; Bone and Bones; Bone Remodeling; Bone Resorption; Cell Survival; Interleukin-17; Interleukins; Macrophage Colony-Stimulating Factor; Mice; Naphthoquinones; Neovascularization, Physiologic; Osteoclasts; Osteogenesis; Osteoporosis; RANK Ligand; RAW 264.7 Cells; Th17 Cells

Non-small cell lung cancer (NSCLC) is one of the most frequently diagnosed cancers and the leading causes of cancer death worldwide. Therefore, new therapeutic agents are urgently needed to improve patient outcomes. Plumbagin (PLB), a natural sesquiterpene present in many Chinese herbal medicines, has been reported for its anti-cancer activity in various cancer cells. In this study, the effects and underlying mechanisms of PLB on the tumorigenesis of NSCLC were investigated. PLB dose-dependently inhibited the growth of NSCLC cell lines. PLB promoted ROS production, activated the endoplasmic reticulum (ER) stress pathway, and induced cell apoptosis, accompanied by the decreased expression level of ADP-ribosylation factor 1 (ARF1) in NSCLC cancer cells, and those effects of PLB could be reversed by the pretreatment with N-acetyl-L-cysteine (NAC). More importantly, the calcium chelator (BM) significantly reversed PLB-induced cell apoptosis. Furthermore, PLB significantly inhibited the growth of both H1975 xenograft and LLC1 tumors and exhibited antitumor activity by enhancing the number and the effector function of CD8

Topics: ADP-Ribosylation Factor 1; Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Down-Regulation; Female; Lung Neoplasms; Lymphocyte Activation; Mice, Nude; Naphthoquinones; Neoplasm Transplantation

Proliferative vitreoretinopathy (PVR) is a serious ophthalmic disease and characterized by the formation of proliferative membranes by retinal pigment epithelial (RPE) cells. In PVR, the contraction and traction of the fibrocellular membranes cause retinal detachment, which can cause reduction surgery for retinal detachment to fail. Fibroblast growth factor-2 (FGF-2) causes RPE cells to form extracellular matrix (ECM), promotes chemotaxis, mitosis, and positively promotes the disease process of PVR. Plumbagin (PLB) is a plant small molecule naphthoquinone compound. It has the functions in anti-tumor, anti-inflammatory, inhibit proliferation. We tried to investigate the possible effects of PLB on the biological behavior of ARPE-19 cells induced by FGF-2 and its underlying mechanisms. Our study confirmed that proliferation, migration, and invasion of ARPE-19 cells induced by FGF-2 (10 ng/ml) were significantly inhibited by PLB. PLB also significantly inhibits the expression of MMP-2/-9, collagen I Alpha 1 (Col1A1), collagen IV Alpha 1 (Col4A1), collagen VI Alpha 1 (Col6A1), and the phosphorylation of FGF receptor (FGFR)-1, FGFR-2, ERK, p38, JNK of FGF-2-induced ARPE-19 cells. In summary, PLB inhibits FGF-2-stimulated proliferation, migration, and invasion of ARPE-19 cells, which may take place through inhibiting the expression of MMP-2/-9, Col1A1, Col4A1, Col6A1, and the mitogen-activated protein kinase (MAPK) pathway. PLB may have a preventive effect on proliferation, migration, and invasion of FGF-2-induced ARPE-19 cells.

Topics: Cell Line; Cell Movement; Cell Proliferation; Collagen; Dipeptides; Epithelial Cells; Fibroblast Growth Factor 2; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthoquinones; Phenylurea Compounds; Pyrimidines; Receptors, Fibroblast Growth Factor; Retinal Pigment Epithelium; RNA, Messenger

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which

Topics: Anti-Bacterial Agents; Carnivorous Plant; Drug Resistance, Bacterial; Metal Nanoparticles; Microbial Sensitivity Tests; Naphthoquinones; Plant Extracts; Pseudomonas aeruginosa; Secondary Metabolism; Silver; Spectrophotometry, Ultraviolet

Hyaluronic acid (HA)-CD44 pathway showed association with several malignancies. The natural polyphenols Plumbagin, Pongapin and Karanjin showed anti-cancer activities in different tumors including cervical carcinoma. To understand their mechanism of anti-cancer activity, the effect of the compounds on HA-CD44 pathway was analyzed in cervical cancer cell line HeLa. The mRNA expression of three different isoforms of CD44 i.e., CD44s, CD44v3, and CD44v6, was differentially downregulated by the compounds. This was validated by Western blot and immunocytochemical analysis of CD44s.The low molecular weight HA (LMW-HA) showed growth promoting activity in HeLa at low concentration, whereas high molecular weight HA (HMW-HA) had no such effect. The compounds could preferentially downregulate the LMW-HA level in HeLa, as evident in the cell as well as in the cell-free conditioned medium. Concentration-dependent upregulation of HA synthase-2 (HAS2) was seen in the cell by the compounds, whereas differential downregulation of hyalurinidases 1-4 (HYAL 1-4), predominantly HYAL1, were seen. The compounds could also downregulate the downstream target of the pathway p-AKT (T-308) in concentration-dependent manner. Thus, the compounds could attenuate the HA-CD44 pathway in HeLa cell to restrict the tumor growth.

Topics: Benzopyrans; Down-Regulation; Female; Flavones; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Hyaluronan Receptors; Hyaluronic Acid; Naphthoquinones; Neoplasm Proteins; Signal Transduction; Uterine Cervical Neoplasms

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant

Topics: Antineoplastic Agents, Phytogenic; Binding Sites; Biological Products; Biosynthetic Pathways; Catalytic Domain; Dihydroorotase; Enzyme Inhibitors; Models, Molecular; Molecular Conformation; Molecular Structure; Mutation; Naphthoquinones; Protein Binding; Pyrimidines; Structure-Activity Relationship

Different aspects of reproductive functions are regulated by mitochondrial-controlled events. This study investigated the effect of plumbagin (PL) on testicular mitochondria with a view to unravelling the mechanism of the antifertility potential of plumbagin in testis of healthy rats. Thirty-two male Wistar strain albino rats were randomly allocated into four groups of eight animals each. The control or healthy group received orally 0.1 % DMSO while animals in the remaining three groups received 2.5 mg PL/kg bdwt, 5.0 mg PL/kg bdwt and 10 mg PL/kg bdwt, respectively, for 14 days. In study two, twenty-four male Wistar rats were randomly divided into three (3) groups and were orally administered 0.1% DMSO (control), 30 and 100 mg/kg PL, respectively once daily for 72 h. Rat testis mitochondria were isolated using differential centrifugation. The mitochondrial Permeability Transition (mPT) pore, mitochondrial ATPase (mATPase) activity and mitochondrial lipid peroxidation were assessed spectrophotometrically. Expression of apoptotic proteins (p53, Bax, Bcl-2) and the release of cytochrome c were determined by immunochemical technique. Reproductive receptors (FSH, PR), the expression of aromatase, Testis Specific Kinase-1 {TESK-1} were quantified by RT-PCR. The various doses of plumbagin (2.5, 5.0 and 10 mg/kg bdwt) induced opening of the testicular mPT pore by 2, 5 and 8 folds, respectively, after 14 days of oral administration. These doses of plumbagin also caused enhancement of mATPase activity, elevated generation of mLPO as well as increases in the concentrations of caspases 9 and 3. Sperm analysis revealed that these doses of PL also caused significant decreases in sperm count and motility and increased sperm abnormalities compared to control. Interestingly, these effects were accompanied by dose-dependent expressions of the Bak, p53 and cytochrome c release. Conversely, the abundance of anti-apoptotic Bcl-2 protein decreased relative to control. The levels of transcripts of FSH and progesterone receptors as well as TESK-1 and aromatase decreased significantly relative to control. Furthermore, PL strongly inhibited p53-MDM2 compared to control. Altogether, these findings show that plumbagin damages testicular cells through the activation of mitochondrial pathway involving the p53 protein network.

Topics: Adenosine Triphosphatases; Animals; Apoptosis; Caspase 9; Cell Death; Lipid Peroxidation; Male; Mitochondria; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Signal Transduction; Spermatozoa; Testis; Tumor Suppressor Protein p53

The biological consequences associated with the conversion of soluble proteins into insoluble toxic amyloids are not only limited to the onset of neurodegenerative diseases but also to the potential health risks associated with supplements of protein therapeutic agents as well. Hence, finding inhibitors against amyloid formation is important, and natural product-based anti-amyloid compounds have gained much interest because of their higher efficacy and biocompatibility. Plumbagin has been identified as a potential natural product with multiple medical benefits; however, it remains largely unclear whether plumbagin can act against amyloid formation of proteins. Here, we show that plumbagin can effectively inhibit the temperature-induced amyloid aggregation of important proteins (insulin and serum albumin). Both experimental and computational data revealed that the presence of plumbagin in protein solutions, under aggregating conditions, promotes a direct protein-plumbagin interaction, which is predominantly stabilized by stronger H-bonds and hydrophobic interactions. Plumbagin-mediated retention of the native structures of proteins appears to play a crucial role in preventing their conversion into insoluble β-sheet-rich amyloid aggregates. More importantly, the addition of plumbagin into a suspension of protein fibrils triggered their spontaneous disassembly, promoting the release of soluble proteins. The results highlight that a possible synergistic effect via both the stabilization of protein structures and the restriction of the monomer recruitment at the fibril growth sites could be important for the mechanism of plumbagin's anti-aggregation effect. These findings may inspire the development of plumbagin-based formulations to benefit both the prevention and treatment of amyloid-related health complications.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Naphthoquinones; Protein Aggregates

The global rise in antimicrobial resistance and lack of discovery of new antimicrobials have created serious concerns. Targeting quorum sensing (QS) and biofilms of pathogenic bacteria is considered a promising approach in antimicrobial drug discovery. This study explored the inhibitory effect of plumbagin against biofilms and QS of

Topics: Anti-Bacterial Agents; Biofilms; Chromobacterium; Computer Simulation; Gram-Negative Bacteria; Naphthoquinones; Pseudomonas aeruginosa; Quorum Sensing; Virulence; Virulence Factors

Plumbagin (PLB), an alkaloid obtained from the roots of the plants of Plumbago genus, is an inhibitor of NADPH oxidase 4 (NOX4). This study aimed to investigate the beneficial effect of PLB against oxygen-glucose deprivation/reoxygenation (OGDR)-induced neuroinjury in human SH-SY5Y neuronal cultures. Our results showed that OGD/R stimulated NOX4 protein expression and reactive oxygen species (ROS) production in SH-SY5Y cells, whereas increased 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) production, resulting in the activation of the NLRP3 inflammasome. And PLB pretreatment reduced the ROS production by regulating the expression of NOX4 and downregulated NF-κB signaling which was induced by OGDR. Furthermore, PLB inhibited OGDR induced NLRP3 inflammasome activation but not PARP1. Overall, PLB improved OGDR induced neuroinjury by inhibiting NOX4-derived ROS-activated NLRP3 inflammasome.

Topics: Apoptosis; Brain Ischemia; Cell Hypoxia; Cell Line; Cell Survival; Glucose; Humans; Inflammasomes; Membrane Potential, Mitochondrial; NADPH Oxidase 4; Naphthoquinones; Neurons; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Plant Extracts; Plant Roots; Plumbaginaceae; Reactive Oxygen Species

Plumbago europaea L. is the main source of plumbagin which is a well-known pharmacological active compound. In this investigation, genetically transformed roots of P. europaea were obtained by improving some factors affecting the efficiency of Agrobacterium rhizoigenes-mediated transformation such as explant type, A. rhizoigenes strain, bacterial infection period, co-cultivation period and acetosyringone concentration. The leaf, hypocotyl and stem explants from in vitro grown plantlets were infected with bacterial strains (A4, ATCC15834, MSU440 and A13). The highest transformation rate of 69.3% was achieved after 7-9 days by inoculating A. rhizogenes MSU440 strain onto the 3-week-old stem explants followed by a co-cultivation period of 2 days on a medium containing 100 μM acetosyringone. To investigate the existence of the rolB gene, polymerase chain reaction was carried out using specific primers. Effects of growth media (MS, 1/2 MS, MS-B5 and ½ MS-B5), different sucrose concentrations and illumination on biomass production and plumbagin biosynthesis in P. europaea hairy root cultures were analyzed using stem explants after infection with MSU440 strain. ½ MS-B5 liquid medium containing 30 g L

Topics: Biomass; Naphthoquinones; Plant Roots; Plants, Genetically Modified; Plumbaginaceae

PLB is a natural naphthoquinone compound isolated from the roots of Plumbago indica plant. Our previous study reported the inhibitory effect of Plumbagin (PLB) on human endocrine resistant breast cancer cell growth and cell invasion.. Since PLB is a naphthoquinone compound, it can be reduced by the cytosolic NADPH: quinone oxidoreductase 1 (NQO1) enzyme. NQO1 expression is upregulated in various types of aggressive cancer including breast cancer. This study investigated the impact of NQO1 on anti-cancer effects of PLB in endocrine-resistant breast cancer cells.. This study was an in vitro study using ER-positive cell line (MCF7) and endocrine-resistant cell lines (MCF7/LCC2 and MCF7/LCC9 cells).. The roles of NQO1 in anti-cancer activity of PLB were investigated by using NQO1 knockdown cells, NQO1 inhibitor and NQO1 overexpressed cells. To study the impact of NQO1 on the effects of PLB on cell viability, apoptosis, invasion and generation of ROS, the following assays were used: MTT assays, annexin V-PE/7-ADD staining flow cytometry, matrigel invasion assays and DCFHDA assays. To study the mechanism of how NQO1 mediated PLB effects in tamoxifen response and apoptosis, we assessed the levels of mRNA expression by using qRT-PCR.. 1. In this study, NQO1 was upregulated in endocrine-resistant cells. 2. PLB did not change the expression of NQO1 but it was able to increase NQO1 activity. 3. The inhibitory effects of PLB on cell proliferation, cell invasion and expression of tamoxifen resistant gene were attenuated in NQO1 knockdown cells or in the presence of NQO1 inhibitor. 4. The effects of PLB to induce apoptosis and generate ROS were also decreased when NQO1 activity was inhibited or when the NQO1 expression was reduced. 5. The anti-cancer effects of PLB increased when NQO1 was upregulated.. The effects of PLB in endocrine-resistant breast cancer cells is dependent on NQO1's activity.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Female; Humans; MCF-7 Cells; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Neoplasm Invasiveness; Tamoxifen

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Humans; MCF-7 Cells; Naphthoquinones; Nuclear Receptor Coactivator 3; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Estrogen; Signal Transduction

Plumbagin, a natural naphthoquinone from the officinal leadwort, has recently been shown to exert promising anti-cancer effects. However, its therapeutic use is hampered by its failure to specifically reach tumors after intravenous administration, without secondary effects on normal tissues. Its poor solubility in water and rapid elimination following

Topics: Animals; Cell Line, Tumor; Mice; Nanoparticles; Naphthoquinones; Transferrin

Studies that evaluated the mechanisms of action of Plumbagin (PLB) and its toxicity may contribute to future therapeutic applications of this compound. We investigate biomarker important in the mechanisms of action correlate the expression of mRNA with the cytotoxic and genotoxic effects of PLB on HepG2/C3A. In the analysis of cytotoxicity, PLB decreased cell viability and membrane integrity at concentrations ≥ 15μM. Xenobiotic-metabolizing system showed strong mRNA induction of CYP1A1, CYP1A2, and CYP3A4, suggesting extensive metabolization. PLB induced apoptosis and an increase in the mRNA expression of genes BBC3, CASP3, and CASP8. At a concentration of 15μM, there was a reduction in the expression of PARP1 mRNA and an increase in the expression of BECN1 mRNA, suggesting that PLB may also induce cell death by autophagy. PLB induced an arrest at the G2/M phase due to DNA damage, as observed in the comet assay. This damage is associated with the increased mRNA expression of genes p21, GADD45A, and H2AFX and with changes in the expression of proteins H2AX, p21, p53, Chk1, and Chk2. These results allow a better understanding of the cellular action of PLB and of its toxicity, thereby contributing to the development of PLB-based drugs, with markers of mRNA expression possibly playing a role as indicators for monitoring toxicity in human cells.

Topics: Antinematodal Agents; Cell Survival; Comet Assay; DNA Damage; Down-Regulation; Hep G2 Cells; Humans; Naphthoquinones; RNA, Messenger

Melanoma is the most dangerous form of skin cancer with a very poor prognosis. Melanoma develops when unrepaired DNA damage causes to skin cells to multiply and form malignant tumors. The current therapy is limited by the highly ability of this disease to metastasize rapidly. Plumbagin is a naphthoquinone (5-hydroxy-2-methyl-1, 4-naphthoquinone), isolated from the roots of medicinal plant Plumbago zeylanica, and it is widely present in Lawsonia inermis L. It has been shown that plumbagin has an anti-proliferative and anti-invasive activities in various cancer cell lines; however, the anti-cancer and anti-metastatic effects of plumbagin are largely unknown against melanoma cells. In this study, we evaluated the effect of plumbagin on B16F10 murine melanoma cells . Plumbagin decreased B16F10 cell viability as well as the cell migration, adhesion, and invasion. The molecular mechanism was studied, and plumbagin downregulated genes relevant in MAPK pathway, matrix metalloproteinases (MMP's), and cell adhesion. Furthermore, plumbagin elevated the expression of apoptosis and tumors suppressor genes, and genes significant in reactive oxygen species (ROS) response. Taken together, our findings suggest that plumbagin has an anti-invasion and anti-metastasis effect on melanoma cancer cells by acting on MAPK pathway and its related genes.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Melanoma; Melanoma, Experimental; Mice; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Plant Extracts; Signal Transduction; Wound Healing

Plumbagin derived from the plant Plumbago indica, known as Chitrak in India, is an example of a medicinal compound used traditionally to cure a variety of ailments. Previous reports have indicated that it can inhibit the growth of Mycobacterium tuberculosis (Mtb), the causative agent of the deadly disease TB. In this investigation, we provide an insight into its mode of action. We show here that a significant mycobacterial target that is inhibited by plumbagin is the enzyme ThyX, a form of thymidylate synthase, that is responsible for the synthesis of dTMP from dUMP in various bacterial pathogens, including Mtb. Using a purified preparation of the recombinant version of Mtb ThyX, we demonstrate that plumbagin, a 2,4 napthoquinone, but not lawsone, a structurally related medicinal compound, inhibits its activity in vitro. We also show that the intracellular [dTTP]/[dATP] ratio in Mycobacterium smegmatis (Msm) cells decrease upon treatment with plumbagin, and this, in turn, leads to cell death. Such a conclusion is supported by the observation that over-expression of thyx in the plumbagin treated Msm cells leads to the restoration of viability. The results of our investigation indicate that plumbagin kills mycobacterial cells primarily by targeting ThyX, a vital enzyme required for their survival.

Topics: Antitubercular Agents; Biological Products; Cell Survival; Deoxyadenine Nucleotides; Mycobacterium tuberculosis; Naphthoquinones; Thymidylate Synthase; Thymine Nucleotides

Prognosis of pancreatic cancer patients remains extremely poor thus, the need for the development of new therapeutic options is crucial. Plumbagin, a naphthoquinone derivative from Plumbago indica has been found to possess various pharmacological properties including anticancer activity. The present study was designed to investigate the inhibitory potential of plumbagin and associated mechanisms in pancreatic cancer cells. Fluorescence and flow cytometric analysis exhibited an increased percentage of apoptotic cells in both monolayer culture and 3D tumor spheroids. Upon plumbagin treatment, reactive oxygen species content of the cancer cells escalated and prompted alleviation of the mitochondrial membrane potential, which triggers caspase-dependent apoptosis. Interestingly, N-acetylcysteine inhibited the plumbagin induced apoptosis. We also found that the expression of Bcl-2 protein decreased and the expression of Bax protein increased. Moreover, plumbagin treatment led to upregulation of cleaved caspase-3 and caspase-9. These results support the views that plumbagin induced stress signals by damaging mitochondria and induce ROS mediated apoptosis via intrinsic apoptotic signaling in pancreatic cancer cells. To summarize, our study suggests that plumbagin may be utilized as a future anti-cancer therapy agent against pancreatic cancer, which is a major threat owing to its stubborn intransigence towards current treatment regimens.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; DNA Damage; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Oxidative Stress; Pancreatic Neoplasms; Reactive Oxygen Species; Signal Transduction

Topics: A549 Cells; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 9; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Naphthoquinones; Reactive Oxygen Species; Structure-Activity Relationship; Tumor Cells, Cultured

Despite the plethora of significant research progress made to develop novel strategies for the treatment of prostate cancer, this disease remains one of the major global health challenges among men. However, using a co-treatment approach utilizing two or more anticancer drugs has shown tremendous success in the treatment of many cancer types. Nanoliposomes are well known to encapsulate multiple drugs and deliver them at the desired site. In this work, we report the synthesis of nanoliposomes (∼100 nm) encapsulating two drugs, plumbagin, and genistein, to synergistically inhibit the growth of prostate cancer cells. The combination of plumbagin and genistein drugs was found inhibiting xenograft prostate tumor growth by ∼80 % without any appreciable toxicity. Mechanistically, the combination of plumbagin and genistein containing nanoliposomes leads to the inhibition of PI3K/AKT3 signaling pathway as well as the decreased population of Glut-1 transporters to impart the retardation in tumor growth. Decrease in proliferative cells and blood vessels are early biological processes that laid the foundation of the observed anti-tumor effect. Thus, a novel, and non-toxic liposomal formulation, containing plumbagin and genistein drugs, is reported, which can deliver anticancer agents to prostate tumors and inhibit the growth.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Cells, Cultured; Drug Delivery Systems; Drug Screening Assays, Antitumor; Genistein; Glucose Transporter Type 1; Humans; Liposomes; Male; Mice; Naphthoquinones; Neoplasms, Experimental; Particle Size; PC-3 Cells; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Surface Properties; Wound Healing

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial lung disease with a poor prognosis similar to that of malignancy. The causes of IPF are not clearly known, and there is no effective therapy to date. In this study, the natural compound plumbagin, which was isolated from

Topics: Animals; Bleomycin; Cell Line; Fibroblasts; Lung; Mice; Naphthoquinones; p300-CBP Transcription Factors; Plant Roots; Plumbaginaceae; Pulmonary Fibrosis

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a small molecule with potent anticancer activity. Like other 1,4-naphthoquinones, it exhibits electrophilic reactivity towards biological nucleophiles. We demonstrate that plumbagin and structurally related 1,4-naphthoquinones with at least one unsubstituted quinoid carbon (C2 or C3) bind to albumin, an ubiquitously present nucleophile, with minimum recovery of free drug. Extraction recovery of plumbagin from albumin in solution showed one-phase exponential decline with a half-live of 9.3 min at 10 μmol/L. In the presence of albumin, plumbagin exhibited instant changes in UV/Vis absorption bands. Electrochemical analysis using cyclic voltammetry showed a decrease in redox peak currents over time until electro-inactivity, thus suggesting the formation of a supramolecular adduct inaccessible for electron transfer. The adduct inhibited cell growth and caused cell-cycle arrest of prostate cancer cells, in part by decreasing levels of the cell-cycle regulator RBBP. The conjugate displayed similar cellular effects to those described for plumbagin, such as decreased levels of androgen receptor and protein kinase C epsilon. The effect of plumbagin-albumin on cancer cells was species-specific, suggesting a receptor-mediated mechanism. Furthermore, it was blocked by cathepsin inhibitor pepstatin A, indicating that lysosomal degradation is involved in the processing of plumbagin-albumin adduct. The spontaneously formed adduct of plumbagin with serum albumin is likely to mediate the biological activities of plumbagin in vivo and to fundamentally influence its pharmacodynamics.

Topics: Animals; Antineoplastic Agents, Phytogenic; Binding Sites; Cell Cycle Checkpoints; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Electrochemical Techniques; Humans; Mice; Molecular Structure; Naphthoquinones; Serum Albumin, Bovine; Signal Transduction; Structure-Activity Relationship; Tumor Cells, Cultured

Plumbagin (PL) pharmacologically plays the anti-proliferative effects in cancer cells, including effective suppression of colorectal cancer (CRC). However, the exact molecular mechanism of PL to treat CRC remains unclear. Using available SwissTargetPrediction and SuperPred databases, the anti-cancer biotargets of PL were identified, and the CRC-diseased targets were obtained through a DisGeNET database. The biological processes, and signaling pathways of PL to treat CRC were identified and visualized. Further, clinical and cell culture data were used to validate some bioinformatic findings. As shown in bioinformatics findings, 64 predictive biotargets of PL to treat CRC were collected, and 7 most important biotargets of tumor protein p53 (TP53), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mitogen-activated protein kinase 1 (MAPK1), E1A-associated protein p300 (EP300), poly (ADP-ribose) polymerase 1 (PARP1), nuclear factor kappa p65 protein (RELA), Bcl-2 like protein 1 (BCL2L1) were identified respectively. In addition, top 20 functional biological processes, signaling pathways of PL to treat CRC were screened and prioritized. In human study, CRC samples showed elevated expressions of neoplastic MAPK1, PARP1 mRNAs and reduced EP300 mRNA level. In cell culture study, PL-treated CRC cells resulted in down-regulated MAPK1, PARP1 mRNA expressions and up-regulation of EP300 mRNA level, characterized with suppressed cell proliferation. Taken together, the therapeutic biotargets and molecular mechanisms of PL to treat CRC were screened and identified by using a systematic pharmacology analysis, and some bioinformatic findings were validated in clinical and cell line experiments. Potentially, these hub biotargets may be the biomarkers for CRC detection and treatment.

Topics: Aged; Antineoplastic Agents, Phytogenic; Cell Proliferation; Colorectal Neoplasms; Databases, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; HCT116 Cells; Humans; Male; Middle Aged; Naphthoquinones; Protein Interaction Maps; Signal Transduction; Systems Biology; Transcriptome

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Survival; Head and Neck Neoplasms; HeLa Cells; Humans; Mice; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; PC-3 Cells; Squamous Cell Carcinoma of Head and Neck; Xenograft Model Antitumor Assays

Cardiovascular disease created enormous health and economic burdens worldwide, which is responsible for the highest mobility and mortality that results in nearly 6.2% of in-hospital deaths every year. Plumbagin is a major bioactive compound that occurs in the Plumbago indica and P. zeylanica with numerous therapeutic benefits. The current research exploration was planned to investigate the therapeutic role of plumbagin against doxorubicin stimulated cardiotoxicity in rats. The cardiotoxicity was stimulated to the rats by administering the 2.5 mg/kg of doxorubicin for 14 days with concurrent supplementation with plumbagin. The hemodynamic parameters were studied by using the tail-cuff plethysmography. The lipid peroxidation and antioxidant status was examined by the standard procedures. The myocardial function and damage markers were assessed with the help of commercial kits. The expression status of inflammatory markers and PI3K/Akt signaling markers were investigated by reverse transcription polymerase chain reaction (RT-PCR) and western blotting analysis, respectively. The plumbagin supplementation appreciably regained the body weight and heart weight of the investigational animals. Hemodynamic parameters and antioxidants statuses were escalated by the plumbagin treatment. The severe elevation in the cardiac damage markers and inflammatory markers were noticeably ameliorated by the plumbagin treatment. The plumbagin treatment also assuaged the overexpression of inflammatory and apoptotic proteins in the heart tissues of doxorubicin-challenged rats. The histopathological analysis revealed that the plumbagin appreciably protected the heart tissues from the doxorubicin-induced damages. The findings of this exploration evidenced that plumbagin treatment attenuated the doxorubicin-stimulated cardiotoxicity in rats.

Topics: Animals; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Biomarkers; Cardiotoxicity; Cytokines; Doxorubicin; Heart; Lipid Peroxidation; Male; Myocardium; Naphthoquinones; Phosphatidylinositol 3-Kinases; Rats; Rats, Wistar; Signal Transduction

Based on the structure of signal transducer and activator of transcription 3 (STAT3), a series of 1,4-naphthoquinones derived from plumbagin (PL) with STAT3 inhibition potential were designed, synthesized, and biologically evaluated in vitro against several human cancer cell lines (MDA-MB-231, HepG2 and A549 cells) and three normal cells. The structure-activity relationship (SAR) and molecular docking result showed that the presence of hydroxyl group at C-5 of PL might interact with STAT3 in the form of hydrogen bonds, which is conducive to the binding of this kind structures with STAT3. Among the target compounds, 7a displayed the most potent inhibition against cancer cells and weaker cytotoxicity on normal cells than PL. The western bolting analysis showed that 7a could suppress the phosphorylation of STAT3 as well as the downstream genes instead of affecting its upstream tyrosine kinases (Src and JAK2) levels and p-STAT1 expression. Furthermore, molecular docking indicated that 7a bound to STAT3 more tightly than PL, and it could significantly induce the apoptosis of cancer cells in vitro. All these results may provide reference for the discovery of effective STAT3 inhibitors.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Humans; Models, Molecular; Molecular Structure; Naphthoquinones; STAT3 Transcription Factor; Structure-Activity Relationship

Plumbagin is a pharmacologically active naphthoquinone present in the Plumbago zeylanica L. having important medicinal properties. The root of P. zeylanica is rich and primary tissue of the plumbagin biosynthesis and accumulation. The complete biosynthetic pathway of plumbagin in plant is still obscure. The present study attempts to understand the plumbagin biosynthetic pathway with the help of differential transcriptome and metabolome analysis of P. zeylanica leaf and root. The transcriptome data showed co-expression of Aldo-keto reductase (PzAKR), Polyketide cyclase (Pzcyclase) and Cytochrome P450 (PzCYPs) transcripts along with the Polyketide synthase (PzPKS) transcripts. Their higher expression in root as compared to leaf supports their possible involvement in plumbagin biosynthesis. The metabolome data of leaf and root revealed naphthalene derivative isoshinanolone that could be potential precursor of plumbagin. Pathway elucidation and transcriptome data of P. zeylanica, will enable and accelerate research on naphthoquinone biosynthesis in plants.

Topics: Gene Expression Regulation, Plant; Genes, Plant; India; Metabolic Networks and Pathways; Metabolome; Naphthoquinones; Plant Leaves; Plant Roots; Plumbaginaceae; Transcriptome

Plumbagin (PL) has been shown to effectively inhibit tumor growth and migration of hepatocellular carcinoma cells in previous studies, but the specific mechanism for this remains unclear. The purpose of this study was to investigate the effects of PL-induced apoptosis in epithelial-mesenchymal transition (EMT) of human hepatocellular carcinoma (HCC) in vivo and in vitro.. SMMC-7721 cells were cultured, an EMT model was induced in vitro by TGF-β1, cell proliferation was detected by the MTT assay, cell invasion was analyzed by the Transwell invasion assay, and the apoptosis rate was measured by flow cytometry. RT-PCR was used to detect vimentin, E-cadherin, N-cadherin and snail mRNA, and Western blotting was used to detect the vimentin, caspase-3, PARP-1, E-cadherin, N-cadherin and snail protein expression levels. HE staining and TUNEL staining, immunohistochemistry and immunofluorescence were used to detect the expression levels of bax and bcl-2 in hepatocarcinoma xenografts and to evaluate their apoptosis in vivo.. The in vitro results showed that PL inhibited the proliferation of EMT model cells, increased the apoptosis rate of the EMT model, and significantly decreased the vimentin, PARP-1, N-cadherin and snail protein levels, but significantly increased E-cadherin and caspase-3 protein expression. In addition, the in vivo results indicated that PL can affect the expression of bax/bcl-2 apoptotic marker proteins.. PL may induce apoptosis of human hepatocellular carcinoma cells undergoing epithelial-mesenchymal transition by increasing the caspase-3 protein level and cleaving vimentin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Signal Transduction; Vimentin

BACKGROUND Anti-angiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). MATERIAL AND METHODS We assessed cellular proliferation, invasion, and activation of growth factors (VEGF and IL-8) with SDF-1 induced in the hepatocellular carcinoma cell line SMMC-7721, and this progression was limited by plumbagin (PL). The human umbilical vein endothelial cell line HUVEC was co-cultured with SDF-1-induced SMMC-7721, and the expressions of CXCR7, CXCR4, and PI3K/Akt pathways after PL treatment were detected by RT-PCR and Western blot analysis. RESULTS The treatment of the hepatoma cell line SMMC-7721 with SDF-1 resulted in enhanced secretion of the angiogenic factors, IL-8 and VEGF, and shows that these stimulatory effects are abolished by PL. The study further demonstrated that PL not only abolishes SDF-1-induced formation of endothelial tubes, but also inhibits expression of CXCR4 and CXCR7, and partially prevents activation of angiogenic signaling pathways. CONCLUSIONS The effect of PL on the SDF-1-CXCR4/CXCR7 axis has become an attractive target for inhibiting angiogenesis in hepatoma cells. Our results provide more evidence for the clinical application of PL as part of traditional Chinese medicine in modern cancer treatment.

Topics: Angiogenesis Inhibitors; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CXCL12; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Liver; Liver Neoplasms; Naphthoquinones; Neoplasm Invasiveness; Neovascularization, Pathologic; Receptors, CXCR; Receptors, CXCR4; Signal Transduction; Stromal Cells; Vascular Endothelial Growth Factor A

Plumbagin (PLB) is a phytochemical being used for centuries in traditional medicines. Recently, its capacity to inhibit the development of human tumors has been observed, through the induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis and metastasis. Here we evaluated the mechanism of action of PLB in the kidney adenocarcinoma 786-O cell line, which are metabolizing cells important for toxicology studies. After the treatment with PLB, we observed increased apoptosis and cell cycle arrest in S and G2/M phases, starting at 5 µM. In addition, PLB was cytotoxic, genotoxic and induced loss of cell membrane integrity. Regarding gene expression, treatment with 7.5 µM PLB reduced the amount of MTOR, BCL2 and ATM transcripts, and increased CDKN1A (p21) transcripts. Phosphorylation levels of yH2AX was increased and MDM2 protein level was reduced following the treatment with PLB, demonstrating its genotoxic effect. Our results suggest that PLB acts in molecular pathways related to the control of proliferation and cell death in 786-O cells.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Autophagy; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Kidney Neoplasms; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phytochemicals; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

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

Radiation induced damage to normal cells is a major shortcoming of conventional radiotherapy, which necessitates the development of novel radio-protective drugs. An ideal radio-modulator would protect normal cells while having cytotoxic effects on cancer cells. Plumbagin is a potent anti-tumour agent and has been shown to sensitize tumour cells to radiation-induced damage. In the present study, we have evaluated the radio-protective potential of plumbagin and found that it protected normal lymphocytes against radiation-induced apoptosis, but did not protect cancer cells against radiation. Plumbagin offered radioprotection even when it was added to cells after irradiation. The ability of only thiol based antioxidants to abrogate the radio-protective effects of plumbagin suggested a pivotal role of thiol groups in the radio-protective activity of plumbagin. Further, protein interaction network (PIN) analysis was used to predict the molecular targets of plumbagin. Based on the inputs from plumbagin's PIN and in light of its well-documented ability to modulate thiol groups, we proposed that plumbagin may act via modulation of caspase enzyme which harbours a critical catalytic cysteine. Indeed, plumbagin suppressed radiation-induced increase in homogenous caspase and caspase-3 activity in lymphocytes. Plumbagin also inhibited the activity of recombinant caspase-3 and mass spectrometric analysis revealed that plumbagin covalently interacts with caspase-3. Further, the in vivo radioprotective efficacy of plumbagin (single dose of 2mg/kg body weight) was demonstrated by its ability to rescue mice against radiation (7.5 Gy; Whole Body Irradiation) induced mortality. These results indicate that plumbagin prevents radiation induced apoptosis specifically in normal cells by inhibition of caspase-3 activity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Proliferation; Gamma Rays; Gene Expression Regulation, Enzymologic; Humans; Lung Neoplasms; Lymphocytes; Mice; Naphthoquinones; Oxidation-Reduction; Phosphorylation; Radiation-Protective Agents

Topics: Antineoplastic Agents; cdc25 Phosphatases; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Humans; MAP Kinase Kinase 7; Models, Molecular; Molecular Docking Simulation; Naphthoquinones

Plumbagin is the main pharmacologically active compound of carnivorous plants in the genera Drosera. It possesses various pharmacological activities, including anticancer and antimalarial activities, and is used in traditional medicine. In this study, we reported a sustainable production system of plumbagin by adding sodium acetate and L-alanine as precursors to in vitro cultures of Drosera burmannii Vahl and Drosera indica L. In addition, plumbagin production was reported in the cultures subjected to different color LED lights. The highest plumbagin level (aerial part 14.625 ± 1.007 mg·g

Topics: Alanine; Drosera; Light; Naphthoquinones; Plant Extracts; Plant Growth Regulators; Plant Roots; Sodium Acetate; Time Factors

Fifty percent of advanced stage ER-positive breast cancer patients develop endocrine resistance. Aberrant activation of Wnt/β-catenin is associated with stem-like phenotypes and epithelial-mesenchymal transition (EMT) process which confers resistance to endocrine therapy. Cancer stem-like cells (CSLCs) can be a vital source of proangiogenic factors including fibroblast growth factor 2 (FGF2) which drives angiogenesis and leads to tumor growth and metastasis. Therefore, targeting Wnt and FGF2 may provide effective treatment for endocrine resistant breast cancer. Our previous in vitro study reported that plumbagin (PLB) was a potent anticancer agent and was able to inhibit EMT in endocrine-resistant cells. This study aimed to further investigate the inhibitory effects of PLB on cancer stem-like phenotypes, tumorigenicity and angiogenesis. The results demonstrated Wnt/β-catenin signaling was activated and was able to form mammospheres with increased cancer stem cell markers (ALDH1, NANOG, and OCT4) in endocrine-resistant cells. PLB significantly inhibited colony-forming, mammosphere formation and decreased cancer stem cell markers. The inhibitory effects of PLB on cell proliferation and invasion were mediated by Wnt signaling pathway. PLB also significantly reduced Wnt responsive genes and β-catenin. Moreover, PLB treatment at doses of 2 and 4 mg/kg/day inhibited tumor growth, angiogenesis and metastasis without any adverse effects on body weight and blood coagulation in orthotopic xenograft nude mice. In conclusion, PLB exerted anti-cancer activity and eliminated stem-like properties by attenuating Wnt/β-catenin signaling and FGF2 expression. These findings suggest that PLB could be a promising agent to treat endocrine resistant breast cancer.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Humans; Mammary Neoplasms, Experimental; Mice, Inbred BALB C; Naphthoquinones; Neoplastic Stem Cells; Neovascularization, Pathologic

The stress response adaptability of Mycobacterium tuberculosis (Mtb) is still unresolved. In this study, we ascribe an important function to the MmpS6-MmpL6 (M6) operon in Mtb stress management.. By using a novel promoter probe in a high-throughput unbiased screen, we identified several quinones as potent inducers of the M6 operon in addition to triclosan.. Triclosan and plumbagin effectively altered the intracellular redox potential in Mtb suggestive of oxidative stress in bacteria. Presence of the functional M6 operon correlated with an enhanced ability of clinical strains to survive in the presence of triclosan.. Similar to the addition of a powerful reactive oxygen species-quenching agent such as N-acetyl cysteine in the medium, introduction of the complete M6 operon was sufficient to increase tolerance of the M6- strains to triclosan and plumbagin by effectively ablating the change in intracellular redox potential of Mtb, signifying the importance of this operon in oxidative stress survival in mycobacteria.

Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Humans; Macrophages; Mycobacterium tuberculosis; Naphthoquinones; Operon; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; THP-1 Cells; Triclosan

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Caryophyllales; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Female; Humans; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Reactive Oxygen Species; Tumor Suppressor Protein p53

Plumbagin (PL), an active naphthoquinone compound, has been demonstrated to be a potential anticancer agent. However, the underlying anticancer mechanism is not fully understood. In this study, the human hepatocellular carcinoma (HCC) SMMC-7721 cell line was studied in an in vitro model. The cell proliferation was inhibited by PL in a dose- and time-dependent manner. Electron microscopy, acridine orange staining, and immunofluorescence were used to evaluate autophagosome formation and LC3 protein expression in PL-treated SMMC-7721 cells. Real-time polymerase chain reaction and Western blot showed that PL treatment suppressed the expression of apoptosis and autophagy factors (LC3, Beclin1, Atg7, and Atg5), which are associated with tumor apoptosis and autophagy in SMMC-7721 cells. In the study of in vitro tumor nude mouse models, PL can inhibit tumor growth. Cell apoptosis and autophagy of the transplanted tumors were evaluated by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, and Western blot. In addition, in the in vivo studies of HCC cells, we found that pretreatment with the autophagy inhibitor 3-methyladenine blocked the formation of apoptosis induced by PL. In contrast, administration of the apoptosis inhibitor Z-VAD did not affect PL-induced autophagy. Taken together, our findings strongly suggest that PL is a promising drug with significant antitumor activity in HCC.

Topics: Animals; Apoptosis; Autophagic Cell Death; Carcinoma, Hepatocellular; Cell Line, Tumor; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Xenograft Model Antitumor Assays

The redox state of the endothelial cells plays a key role in the regulation of the angiogenic process. The modulation of the redox state of endothelial cells (ECs) could be a viable target to alter angiogenic response. In the present work, we synthesized a redox modulator by caging 5-hydroxy 2-methyl 1, 4-napthoquinone (Plumbagin) on silver nano framework (PCSN) for tunable reactive oxygen species (ROS) inductive property and tested its role in ECs during angiogenic response in physiological and stimulated conditions. In physiological conditions, the redox modulators induced the angiogenic response by establishing ECs cell-cell contact in tube formation model, chorio allontoic membrane, and aortic ring model. The molecular mechanism of angiogenic response was induced by vascular endothelial growth factor receptor 2 (VEGFR2)/p42-mitogen-activated protein kinase signaling pathway. Under stimulation, by mimicking tumor angiogenic conditions it induced cytotoxicity by generation of excessive ROS and inhibited the angiogenic response by the loss of spatiotemporal regulation of matrix metalloproteases, which prevents the tubular network formation in ECs and poly-ADP ribose modification of VEGF. The mechanism of opposing effects of PCSN was due to modulation of PKM2 enzyme activity, which increased the EC sensitivity to ROS and inhibited EC survival in stimulated condition. In normal conditions, the endogenous reactive states of NOX4 enzyme helped the EC survival. The results indicated that a threshold ROS level exists in ECs that promote angiogenesis and any significant enhancement in its level by redox modulator inhibits angiogenesis. The study provides the cues for the development of redox-based therapeutic molecules to cure the disease-associated aberrant angiogenesis.

Topics: Animals; Chick Embryo; Endothelial Cells; Humans; Nanoparticles; Nanotechnology; Naphthoquinones; Neovascularization, Pathologic; Neovascularization, Physiologic; Oxidation-Reduction; Reactive Oxygen Species; Silver

Chronic consumption of fructose causes obesity and nonalcoholic fatty liver disease (NAFLD). Currently available therapies have limitations; hence there is a need to screen new molecules. Plumbagin is a naphthoquinone present in the roots of Plumbago species which has hypolipidemic and hepatoprotective activities.. Rats were divided into five groups: normal control, disease control, orlistat, plumbagin (0.5 mg/kg and 1 mg/kg body weight). The normal control group received standard diet and drinking water while the remaining groups received fructose in drinking water alongwith the standard diet for 16 weeks. Orlistat and plumbagin were administered orally from the 9. Fructose feeding resulted in a significant increase in the body weight gain, calorie intake, visceral fat, liver weight, blood glucose and insulin and caused dyslipidemia which was mitigated by plumbagin. Plumbagin exerted antioxidant, anti-inflammatory and anti-fibrotic effects in the liver and reduced the hepatic lipids. Plumbagin reduced the gene expression of SREBP-1c and increased that of PPAR-α. Plumbagin reduced the hypertrophy of adipocytes and ameliorated the degenerative changes in the liver.. Plumbagin thus seems to be a promising molecule for the management of obesity and NAFLD.

Topics: Adjuvants, Immunologic; Animals; Antioxidants; Fructose; Inflammation; Lipid Metabolism; Male; Naphthoquinones; Non-alcoholic Fatty Liver Disease; Obesity; Oxidative Stress; Random Allocation; Rats; Rats, Wistar

ERK is a component of mitogen-activated protein kinases that controls a range of cellular processes including cell proliferation and survival. The upregulation of ERK has been associated with apoptosis inhibition in response to various stimuli including chemotherapeutic agents. Research has suggested that the upregulation of ERK signaling by the anticancer agent paclitaxel leads to acquired resistance of cells to this compound. The presented research focused on determining the role of plumbagin, a naturally derived naphthoquinone, in the sensitization of breast cancer cells to paclitaxel-induced cell death and the involvement of ERK signaling in this process. The results of the study indicated that plumbagin increases the sensitivity of breast cancer cells to paclitaxel. Moreover, a synergistic effect between plumbagin and paclitaxel was observed. Plumbagin was shown to decrease levels of phosphorylated ERK in breast cancer cells and abrogated paclitaxel-induced ERK phosphorylation. The role of ERK in plumbagin-mediated sensitization of breast cancer cells to paclitaxel was shown through the enhancement of the synergistic effect between compounds in cells with decreased ERK expression. Furthermore, plumbagin reduced p-ERK levels in paclitaxel-resistant breast cancer cells and resensitized paclitaxel-resistant cells to this compound. These results imply that plumbagin inhibits ERK activation in breast cancer cells, which plays a role in the sensitization of cells to paclitaxel-induced cell death.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Naphthoquinones; Paclitaxel

Allelopathy and bioassays constitute fundamental tools in the search for new herbicide templates. The work described here is a continuation of a previous study focused on the structure-activity relationships between transport phenomena and phytotoxic activity. Different modifications were made to the naphthoquinone backbone and two key factors were identified as being responsible for changes in activity: lipophilicity and the nature of the functional group. The study of other naturally occurring and semi-synthetic naphthoquinones was also proposed.. A total of 12 5-O-acyl plumbagins and 18 analogs with unsaturated and aromatic substituents at positions 2 and 5 were synthesized. These compounds were evaluated in the wheat coleoptile bioassay and against Standard Target Species (STS) and three weeds, namely Echinochloa crus-galli L., Lolium rigidum Gaud. and Lolium perenne L. A strong structure-function relationship was observed for the different naphthoquinones and root and shoot length were the parameters that were most affected.. Strong inhibitory effects were observed for the isomeric forms 23 and 33 and the derivatives with a free hydroxyl group, i.e. 24 and 30, gave values higher than 70% inhibition for root length in barnyardgrass and perennial ryegrass. These results highlight the potential of these compounds as models in the development of herbicides based on natural products. © 2019 Society of Chemical Industry.

Topics: Biological Control Agents; Echinochloa; Herbicides; Lolium; Naphthoquinones; Plant Weeds; Structure-Activity Relationship

Plumbagin (5-hydroxy-2-methyl-1,4-napthoquinone) is a bicyclic naphthoquinone, found in three major plant families viz. Plumbaginaceae, Ebenceae and Droseraceae. The phytochemical is reported to exhibit various pharmacological properties. In this study, plumbagin isolated from Plumbago zeylanica L. was investigated for its in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Against 100 MRSA isolates that included multi-drug-resistant phenotypes, plumbagin showed consistent activity with a narrow minimum inhibitory concentration (MIC) range of 4-8 μg ml

Topics: Anti-Bacterial Agents; Cell Wall; Ciprofloxacin; Cytoplasm; Drug Synergism; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Naphthoquinones; Phytochemicals; Piperacillin; Plant Extracts; Plumbaginaceae

This study was performed to evaluate the antidiabetic and wound healing activity of plumbagin in diabetic rats by macroscopical, biochemical, histological, immunohistochemical and molecular methods. Percentage of wound closure and contraction was delayed in diabetic rats when compared to non-diabetic group. There was significant reduction in period of epithelialization, collagen and protein content. Serum insulin level was significantly lowered together with increase in glucose level in diabetic rats. Lipid levels were increased significantly with concomitant decrease in HDL level. The mRNA levels of Nrf2, collagen-1, TGF-β and α-SMA were significantly lowered whereas Keap-1 levels were increased in diabetic rats. The level of lipid peroxides was increased while the levels of antioxidants were lowered significantly. ELISA results reveal upregulated levels of inflammatory markers. Western blot result shows upregulated levels of CD68 and CD163 proteins in wound area of diabetic rats. Histopathological observation revealed increased inflammatory cells infiltration in diabetic control. Immunofluorescent staining and immunohistochemical analysis also displayed delayed wound healing in diabetic groups. Diabetic rats treated with 10% and 20% plumbagin showed increased epithelialization, collagen deposition, increased serum insulin level and increased antioxidant status. Lipid peroxides and lipid levels were lowered significantly with increase in HDL level. Inflammatory markers were lowered, and growth factors expressions were increased markedly. Thus, the results of the study indicated that plumbagin administration could improve wound healing activity and could serve as a potent antidiabetic and anti-inflammatory agent.

Topics: Animals; Antioxidants; Collagen; Collagen Type I; Diabetes Mellitus, Experimental; Disease Models, Animal; Insulin; Male; Naphthoquinones; Rats; Rats, Wistar; Skin; Wound Healing

The purpose of this study was to investigate plumbagin (PL) on liver fibrosis in vitro and in vivo and to explore the underlying mechanisms.. Carbon tetrachloride (CCl. The results revealed that PL significantly prevented CCl. The results of our study indicate that PL can mitigate liver fibrosis in vitro and in vivo, which may be related to the ROS-mediated NF-кB signaling pathway.

Topics: Animals; Antioxidants; Cell Nucleus; Collagen Type I; Collagen Type III; Hepatic Stellate Cells; Interleukin-1beta; Liver Cirrhosis; Male; NADPH Oxidase 4; Naphthoquinones; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Phosphorylation; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Transcription Factor RelA

Plumbagin exerts effective anti-hepatocellular carcinoma (HCC) benefits, however, the detailed mechanisms behind these effects are not yet completely elucidated. The pharmacological targets and molecular mechanisms of plumbagin against HCC were revealed through conducting network pharmacology approach before experimentative verification.. The web-accessible databases of herbal ingredients' targets (HIT), Swiss-Target-Prediction and Super-Pred were used to predict the therapeutic targets of plumbagin, followed by combined with pathogenic targets of HCC from oncogenomic database of hepatocellular carcinoma (OncoDB.HCC) and Liverome databases to obtain the predominant targets of plumbagin-treating HCC. The database for annotation, visualization and integrated discovery (DAVID) was applied to output the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment by use of all predominant targets for computerized visualization. The validated data of human and cell culture were subjected to a group of medical imaging, biochemical tests and immunostaining, respectively.. As revealed in bioinformatic data, 19 predominant targets of plumbagin-treating HCC were obtained, and 5 top targets of TP53, MAPK1, MAP2K1, RAF1 and CCND1 were the most important biomolecules in anti-HCC effects exerted by plumbagin. Other identifiable 102 GO items were showed, including 66 biological processes, and 12 cellular components, 24 molecular functions. And 67 KEGG pathways were mainly involved in neoplastic signaling. In human data, HCC sections showed increased expressions of hepatocellular TP53, MAPK1, accompanied with positive clinical imaging results for HCC. In plumbagin-treated HepG2 cells, reduced TP53, MAPK1 protein expressions were observed, accompanied with cell arrest and apoptosis.. Collectively, the pharmacological targets and mechanisms of plumbagin-treating HCC were predicted and integrated through the method of network pharmacology, followed by some investigative validations. Interestingly, these 5 predominant biomolecules may be the potential targets for screening and treating HCC.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Computational Biology; Cyclin D1; Female; Hep G2 Cells; Humans; Liver Neoplasms; Male; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Naphthoquinones; Proto-Oncogene Proteins c-raf; Tumor Suppressor Protein p53

1,4-naphthoquinones, especially juglone, are known for their anticancer activity. However, plumbagin, lawsone, and menadione have been less investigated for these properties. Therefore, we aimed to determine the effects of plumbagin, lawsone, and menadione on C6 glioblastoma cell viability, ROS production, and mitochondrial function.. Cell viability was assessed spectrophotometrically using metabolic activity method, and by fluorescent Hoechst/propidium iodide nuclear staining. ROS generation was measured fluorometrically using DCFH-DA. Oxygen uptake rates were recorded by the high-resolution respirometer Oxygraph-2k.. Plumbagin and menadione displayed highly cytotoxic activity on C6 cells (IC. Plumbagin and menadione exhibit strong prooxidant, mitochondrial oxidative phosphorylation uncoupling and cytotoxic activity. In contrast, lawsone demonstrates a moderate effect on C6 cell viability and mitochondrial functions, and possesses strong antioxidant properties.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Cell Line, Tumor; Cell Survival; Glioblastoma; Mitochondria; Naphthoquinones; Oxidants; Oxidation-Reduction; Oxidative Stress; Phosphorylation; Phytotherapy; Plant Extracts; Rats; Reactive Oxygen Species; Uncoupling Agents; Vitamin K 3

BACKGROUND This study aimed to use a network pharmacology approach to establish the effects of plumbagin on pancreatic cancer (PC) and to predict core targets and biological functions, pathways, and mechanisms of action. MATERIAL AND METHODS Genes associated with the pathogenesis of PC were obtained from a database of gene-disease associations (DisGeNET). Putative genes associated with plumbagin were identified from the databases of drug target identification (PharmMapper), target prediction of bioactive components (SwissTargetPrediction), and comprehensive drug target information (DrugBank). PC targets of plumbagin were harvested by using a functional enrichment analysis tool (FunRich). The data of function-related protein-protein interactions (PPIs) with a confidence score >0.9 were obtained by using functional protein association networks (STRING). The network interactions of plumbagin and PC targets and function-related proteins were constructed through complex network analysis and visualization (Cytoscape). The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis were used to identify the effects of plumbagin. RESULTS The most important biotargets for plumbagin in PC were identified as TP53, MAPK1, BCL2, and IL6. A total of 1,731 annotations and 121 enriched pathways for plumbagin and PC were identified by KEGG and GO analysis. The top 10 signaling pathways of plumbagin and PC were screened, followed by identification of biological components and functions. CONCLUSIONS Network pharmacology established the effects of plumbagin on PC, predicted core targets, biological functions, pathways, and mechanisms of action. Further studies are needed to validate these predictive biotargets in PC.

Topics: Biomarkers, Tumor; Computational Biology; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Ontology; Gene Regulatory Networks; Humans; Naphthoquinones; Pancreatic Neoplasms; Protein Interaction Maps; Signal Transduction

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.

Topics: Cell Death; Cell Line; Cell Line, Tumor; Endoplasmic Reticulum Stress; Homeostasis; Humans; Membrane Potential, Mitochondrial; Naphthoquinones; Neoplasms; Proteasome Inhibitors; Sulfhydryl Compounds; Vacuoles

Study of the chemical constituents of the roots of Plumbago zeylanica L. collected in Taiwan led to the isolation and identification of a new naphthoquinone dimer, plumzeylanone (1), along with eight known compounds (2-9). Nine naphthoquinones isolated from this plant were assayed for cell growth inhibition activity using NALM-6 (human B cell precursor leukaemia), A549 (human lung adenocarcinoma), Colo205 (human colorectal adenocarcinoma) and KB (human epidermoid carcinoma). Plumzeylanone (1), a novel plumbagin dimer, suppressed cell proliferation in only NALM-6 cells (IC

Topics: Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dimerization; Humans; Naphthoquinones; Plant Roots; Plumbaginaceae; Taiwan

The purpose of this study was to investigate the clinical and histopathological characteristics of GLUT1 in human tongue squamous cell carcinoma (TSCC) and the role of plumbagin (PLB)-mediating GLUT1 in the growth of TSCC.. Forty-five cases of TSCC samples were collected and the expression and location of GLUT1 was analyzed. The role and mechanism of PLB meditating GLUT1 in the inhibitory growth of human TSCC cell line CAL27 were investigated in vitro and vivo.. The expression of GLUT1 was observed in all samples of human TSCC by immunohistochemical staining. GLUT1 expression was significantly correlated with lymph node metastasis and clinical stage in TSCC. PLB treatment decreased cell viability and colony formation, and increased cell apoptosis in association with the downregulation of GLUT1 via inhibiting PI3K/Akt pathway in vitro and PLB suppressed tumor growth in correlation with downregulation of GLUT1, compared with control group in vivo.. The findings demonstrated a novel anti-cancer mechanism of PLB, inhibitory TSCC growth via suppressing PI3K/Akt/GLUT1 pathway, which will supply a theoretical basis for PLB to treat TSCC.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Female; Glucose Transporter Type 1; Humans; Ki-67 Antigen; Male; Matrix Metalloproteinase 2; Mice; Middle Aged; Naphthoquinones; Platelet Endothelial Cell Adhesion Molecule-1; Tongue Neoplasms; Tumor Stem Cell Assay

Large cell lung cancer (LCLC) patients have a poor prognosis because their tumors are highly malignant and show resistance to chemotherapy and radiotherapy. Plumbagin has anticancer activity toward several tumor types, but its effects on LCLC are unknown. This study investigated the effects of plumbagin on human L9981 and NL9980 large cell lung cancer cells and the mechanisms underlying its action.. After the introduction of exogenous IL-6, the mRNA expression of signaling genes and downstream genes was significantly increased in a concentration-dependent manner. Furthermore, plumbagin significantly inhibited the expression of the above mentioned genes in a dose-dependent and time-dependent manner. The mRNA expression levels of downstream genes were correlated with those of signaling genes.. Plumbagin was found to significantly inhibit the proliferation and invasion of L9981 and NL9980 cells, and may be an effective therapy for LCLC through targeting the IL-6/STAT3 signaling pathway.

Topics: Antineoplastic Agents; Carcinoma, Large Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Interleukin-6; Lung Neoplasms; Naphthoquinones; Phosphorylation; Signal Transduction; STAT3 Transcription Factor

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers, and it requires novel treatment approaches and effective drugs. In the present study, we found that treatment with plumbagin, a natural compound, reduced proliferation and survival of the KYSE150 and KYSE450 ESCC cell lines in a dose-dependent manner in vitro. The drug also effectively inhibited the viability of primary ESCC cells from fresh biopsy specimens. Furthermore, plumbagin-induced mitotic arrest and massive apoptosis in ESCC cells. Notably, the drug significantly suppressed the colony formation capacity of ESCC cells in vitro and the growth of KYSE150 xenograft tumors in vivo. At the molecular level, we found that exposure to plumbagin decreased both polo-like kinase 1 (PLK1) and phosphorylated protein kinase B (p-AKT) expression in both ESCC cell lines. Enforced PLK1 expression in ESCC cells not only markedly rescued cells from plumbagin-induced apoptosis and proliferation inhibition but also restored the impaired AKT activity. Furthermore, signal transducer and activator of transcription 3 (STAT3), a transcription factor of PLK1, was also inactivated in plumbagin-treated ESCC cells; however, the overexpression of a constitutively activated STAT3 mutant, STAT3C, reinstated the plumbagin-elicited blockade of PLK1-AKT signaling in ESCC cells. Taken together, these findings indicate that plumbagin inhibits proliferation and potentiates apoptosis in human ESCC cells in vitro and in vivo. Plumbagin may exert these antitumor effects by abrogating STAT3-PLK1-AKT signaling, which suggests that plumbagin may be a novel, promising anticancer agent for the treatment of ESCC.

Topics: Animals; Apoptosis; Carcinogenesis; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Humans; Mice, Nude; Naphthoquinones; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Topics: Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Cell Line, Tumor; Drug Synergism; Electron Transport; Humans; Mitochondria; Models, Molecular; Molecular Conformation; Naphthoquinones; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Oxygen Consumption; Structure-Activity Relationship

Prostate cancer (PCa) is the second most frequently diagnosed cancer in men worldwide. Currently available therapies for hormone-refractory PCa are only marginally effective. Plumbagin (PLB), a natural naphthoquinone isolated from the traditional folk medicine Plumbago zeylanica, is known to selectively kill tumor cells. Nevertheless, antitumor mechanisms initiated by PLB in cancer cells have not been fully defined.. MTT assay was used to evaluate the effect of PLB on the viability of cancer cells. Cell apoptosis and reactive oxygen species (ROS) production were determined by flow cytometry. Protein expression was detected by western blotting. In vivo anti-tumor effect was measured by using tumor xenoqraft model in nude mice.. In the present study, we found that PLB decreases cancer cell growth and induces apoptosis in DU145 and PC-3 cells. In addition, by increasing intracellular ROS levels, PLB induced a lethal endoplasmic reticulum stress response in PCa cells. Importantly, blockage of ROS production significantly reversed PLB-induced ER stress activation and cell apoptosis. In vivo, we found that PLB inhibits the growth of PCa xenografts without exhibiting toxicity Treatment of mice bearing human PCa xenografts with PLB was also associated with induction of ER stress activation.. Inducing ER stress by PLB thus discloses a previously unrecognized mechanism underlying the biological activity of PLB and provides an in-depth insight into the action of PLB in the treatment of hormone-refractory PCa.

Topics: Acetylcysteine; Activating Transcription Factor 4; Animals; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; eIF-2 Kinase; Endoplasmic Reticulum Stress; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Prostatic Neoplasms; Reactive Oxygen Species; RNA Interference; Transcription Factor CHOP; Transplantation, Heterologous

Treatment of mice harboring PTEN-P2 tumors in the prostate or on prostate tissue in vivo with 5-hydroxy-2-methyl-1,4-naphthoquinone, also known as plumbagin, results in tumor regression in castrated mice, but not in intact mice. This suggested that dihydrotestosterone (DHT) production in the testes may prevent cell death due to plumbagin treatment, but the underlying mechanism is not understood. We performed RNA-seq analysis on cells treated with combinations of plumbagin and DHT, and analyzed differential gene expression, to gain insight into the interactions between androgen and plumbgin. DHT and plumbagin synergize to alter the expression of many genes that are not differentially regulated by either single agent when used alone. These experiments revealed that, for many genes, increases in mRNAs caused by DHT are sharply down-regulated by plumbagin, and that many transcripts change in response to plumbagin in a DHT-dependent manner. This suggests that androgen receptor mediates some of the effects of plumbagin on gene expression.

Topics: Androgens; Animals; Cell Line, Tumor; Dihydrotestosterone; Gene Expression Regulation, Neoplastic; Male; Mice; Naphthoquinones; Prostate; Prostatic Neoplasms; Receptors, Androgen; Testis; Transcriptome

Cytochrome P450 2J2 (CYP2J2) is not only highly expressed in many kinds of human tumors, but also promotes tumor cell growth via regulating the metabolism of arachidonic acids. CYP2J2 inhibitors can significantly reduce proliferation, migration and promote apoptosis of tumor cells by inhibiting epoxyeicosatrienoic acids (EETs) biosynthesis. Therefore screening CYP2J2 inhibitors is a significant way for the development of anti-cancer drug.. The aim of this study was to identify a new CYP2J2 inhibitor from fifty natural compounds obtained from plants.. CYP2J2 inhibitor was screened from a natural compounds library and further the inhibitory manner and mechanism were evaluated. Its cytotoxicity against HepG2 and SMMC-7721 cell lines was also estimated.. The inhibitory effect was evaluated in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant CYP2J2 (rCYP2J2), using astemizole as a probe substrate and inhibitory mechanism was illustrated through molecular docking. The cytotoxicity was detected using SRB.. In all candidates, plumbagin showed the strongest inhibitory effect on the CYP2J2-mediated astemizole O-demethylation activity. Further study revealed that plumbagin potently inhibited CYP2J2 activity with IC. This study found out a new CYP2J2 inhibitor plumbagin from fifty natural compounds. Plumbagin presented a potential of anti-cancer pharmacological activity.

Topics: Animals; Antineoplastic Agents; Biological Products; Carcinoma, Hepatocellular; Cell Proliferation; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Evaluation, Preclinical; Hepatocytes; Humans; Kinetics; Liver Neoplasms; Male; Microsomes, Liver; Molecular Docking Simulation; Naphthoquinones; Rats, Sprague-Dawley

Although plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) protects against cerebral ischemia and spinal cord injury-induced oxidative stress and inflammation by activating the nuclear factor-erythroid 2-related factor-2 /antioxidant response element (Nrf2/ARE) pathway, its role in the amelioration of neurodegenerative diseases remains unexplored. In the present study, we investigated the effect of plumbagin on Alzheimer's disease (AD)-like condition in mice. The animals were treated intracerebroventricularly with streptozotocin (STZ; 3 mg/kg) twice, on day 1 and 3, to induce AD-like condition, and the symptoms were evaluated after 14 days. While the loss of learning and memory performance was evident in the mice subjected to Morris water maze (MWM), there was a striking increase in the population of astrocytes labelled with glial fibrillary acidic protein (GFAP) in the hippocampus. Daily intraperitoneal (i.p.) treatment with plumbagin (0.5 and 1 mg/kg), starting from 1 h prior to first dose of STZ, significantly prevented the cognitive deficits in MWM. On the other hand, administration of Nrf2/ARE pathway inhibitor, trigonelline (10 and 15 mg/kg, i.p.) enhanced the effects of STZ. Pre-treatment with subeffective dose of trigonelline (5 mg/kg) significantly attenuated the effects of plumbagin in MWM. While plumbagin prevented the STZ induced GFAP expression, this effect of plumbagin was attenuated by trigonelline. Moreover, the in silico docking study revealed potent inhibitory effect of plumbagin on β-secretase enzyme. The results of the present study suggest that plumbagin improves cognitive function in STZ induced mouse model of AD possibly via Nrf2/ARE mediated suppression of astrogliosis and inhibition of β-secretase enzyme.

Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Animals; Antioxidant Response Elements; Cerebral Cortex; Cognition Disorders; Cognitive Dysfunction; Disease Models, Animal; Hippocampus; Male; Maze Learning; Memory; Mice; Naphthoquinones; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; Streptozocin

BACKGROUND This study aimed to explore the effects of plumbagin (PLB) on epithelial-to-mesenchymal transition in retinal pigment epithelial (RPE) cells and in proliferative vitreoretinopathy (PVR) rabbit models. MATERIAL AND METHODS Rabbit RPE cells were exposed to various concentrations (0, 5, 15, and 25 µM) of PLB. Motility, migration, and invasion of PLB-treated cells were determined in vitro using Transwell chamber assays and scratch wound assays. The contractile ability was evaluated by cell contraction assay. Expression of matrix metalloproteinases (MMPs) and epithelial-mesenchymal transition (EMT) markers were assessed by western blotting. Furthermore, PLB was injected in rabbit eyes along with RPE cells after gas compression of the vitreous. The presence of PVR was determined by indirect ophthalmoscopy on days 1, 7, 14, and 21 after injection. Also, optical coherence tomography (OCT), ultrasound images, electroretinograms (ERG), and histopathology were used to assess efficacy and toxicity. RESULTS PLB significantly inhibited the migration and invasion of RPE cells. The agent also markedly reduced cell contractive ability. Furthermore, PLB treatment resulted in the decreased expression of MMP-1, MMP2, α-SMA, and the protection of ZO-1. In addition, the PLB-treated eyes showed lower PVR grades than the untreated eyes in rabbit models. PLB exhibited a wide safety margin, indicating no evidence of causing retinal toxicity. CONCLUSIONS PLB effectively inhibited the EMT of rabbit RPE cells in vitro and in the experimental PVR models. The results open new avenues for the use of PLB in prevention and treatment of PVR.

Topics: Animals; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Naphthoquinones; Rabbits; Retinal Pigment Epithelium; Vitreoretinopathy, Proliferative

This study aimed to explore the effects of plumbagin (PLB) on ARPE-19 cells and underlying mechanism.. Cultured ARPE-19 cells were treated with various concentrations (0, 5, 15, and 25 μM) of PLB for 24 h or with 15 μM PLB for 12, 24 and 48 h. Then cell viability was evaluated by MTT assay and DAPI staining, while apoptosis and cell cycle progression of ARPE cells were assessed by flow cytometric analysis. Furthermore, the level of main regulatory proteins was examinated by Western boltting and the expression of relative mRNA was tested by Real-Time PCR.. PLB exhibited potent inducing effects on cell cycle arrest at G2/M phase and apoptosis of ARPE cells via the modulation of Bcl-2 family regulators in a concentration- and time-dependent manner. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways contributing to the anti-proliferative activities in ARPE cells.. This is the first report to show that PLB could inhibit the proliferation of RPE cells through down-regulation of modulatory signaling pathways. The results open new avenues for the use of PLB in prevention and treatment of proliferative vitreoretinopathy.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line; Cell Survival; Drugs, Chinese Herbal; Epithelial Cells; Humans; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Plumbaginaceae; Proto-Oncogene Proteins c-akt; Retinal Pigment Epithelium; Signal Transduction; TOR Serine-Threonine Kinases; Vitreoretinopathy, Proliferative

Plumbagin, a vitamin K3 analogue is the major active constituent in several plants including root of Plumbago indica Linn. This compound has been shown to exhibit a wide spectrum of pharmacological activities. The present investigation was to evaluate the ameliorative effects of plumbagin (PL) against severe malaria pathogenesis due to involvement of oxidative stress and inflammatory response in Plasmodium berghei infected malaria in mice. Malaria pathogenesis was induced by intra-peritoneal injection of P. berghei infected red blood cells into the Swiss albino mice. PL was administered orally at doses of 3, 10 and 30 mg/kg/day following Peter's 4 day suppression test. Oral administration of PL showed significant reduction of parasitaemia and increase in mean survival time. PL treatment is also attributed to significant increase in the blood glucose and haemoglobin level when compared with vehicle-treated infected mice. Significant inhibition in level of oxidative stress and pro-inflammation related markers were observed in PL treated group. The trend of inhibition in oxidative stress markers level after oral treatment of PL was MPO > LPO > ROS in organ injury in P. berghei infected mice. This study showed that plumbagin is able to ameliorate malaria pathogenesis by augmenting anti-oxidative and anti-inflammatory mechanism apart from its effect on reducing parasitaemia and increasing mean survival time of malaria-induced mice.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents; Antimalarials; Disease Models, Animal; Dose-Response Relationship, Drug; Inflammation; Malaria; Male; Mice; Naphthoquinones; Oxidative Stress; Plasmodium berghei; Plumbaginaceae

Topics: 3T3-L1 Cells; Adipocytes; Animals; Cell Differentiation; Humans; Kinetics; Lipase; Male; Mice; Molecular Docking Simulation; Naphthoquinones; Obesity; Plant Roots; Plumbaginaceae; Rats; Rats, Wistar; Triglycerides

High levels of both nitric oxide (NO) and reactive oxygen species (ROS) could act as pro-apoptotic signals in cancerous cells. In this study, we conjugated diazeniumdiolates (NONOates), an important class of NO donors, with a natural occurring plumbagin (PL) which is primarily an excellent ROS inducer. Herein, a total of 12 novel plumbagin/NONOate hybrids have been synthesized and evaluated for their inhibitory effects on a panel of human cancer cell lines (MDA-MB-231, A549, HepG2 and HCT-116 cells) and two normal human cells (HK-2 and WRL-68 cells). Among them, compounds 10a and 10b demonstrated superior potencies compared to their parent compound (IC

Topics: Antineoplastic Agents; Apoptosis; Azo Compounds; Cell Line; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Naphthoquinones; Reactive Oxygen Species; Structure-Activity Relationship

Although plumbagin, a natural naphthoquinone, has exhibited antiproliferative activity in numerous types of cancer, its anticancer potential in esophageal squamous cell carcinoma (ESCC) remains unclear. In the present study, the effect of plumbagin on the growth of ESCC cells was investigated in vitro and in vivo. ESCC cells were treated with plumbagin and tested for cell cycle distribution and apoptosis. The involvement of STAT3 signaling in the effect of plumbagin was examined. The results demonstrated that plumbagin treatment suppressed ESCC cell viability and proliferation, yet normal esophageal epithelial cell viability was not affected. Plumbagin treatment increased the proportion of cells in the G0/G1 phase of the cell cycle and decreased the proportion of cells in the S phase. Furthermore, plumbagin‑treated ESCC cells displayed a significantly greater % of apoptotic cells. Western blot analysis confirmed that plumbagin upregulated tumor protein p53 and cyclin‑dependent kinase inhibitor 1A (also known as p21), while it downregulated cyclin D1, cyclin‑dependent kinase 4, and induced myeloid leukemia cell differentiation protein Mcl‑1. Mechanistically, plumbagin inhibited STAT3 activation, and overexpression of constitutively active STAT3 reversed the plumbagin‑mediated growth suppression in ESCC cells. In vivo studies demonstrated that plumbagin delayed the growth of ESCC xenograft tumors and reduced STAT3 phosphorylation. Overall, plumbagin was demonstrated to target STAT3 signaling and to inhibit the growth of ESCC cells both in vitro and in vivo, suggesting that it may represent a potential anticancer agent for ESCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; G1 Phase; Humans; Mice; Naphthoquinones; Resting Phase, Cell Cycle; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Ischemia-reperfusion (I/R) injury is a pathological process which magnifies with the ensuing inflammatory response and endures with the increase of oxidants especially during reperfusion. The present study was conducted to assess the possible modulatory effects of plumbagin, the active constituent extracted from the roots of traditional medicinal plant Plumbago zeylanica L., on the dire role of high mobility group box 1 (HMGB1) as well as the associated inflammation, oxidative stress and apoptotic cell death following hepatic I/R. Four groups of rats were included: sham-operated, sham-operated treated with plumbagin, I/R (30 min ischemia and 1 h reperfusion) and I/R treated with plumbagin. Pretreatment with plumbagin markedly improved hepatic function and structural integrity compared to the I/R group, as manifested by depressed plasma transaminases and lactate dehydrogenase (LDH) activities as well as alleviated tissue pathological lesions. Plumbagin prominently hampered HMGB1 expression and subsequently quelled inflammatory cascades, as nuclear factor κB (NF-κB), tumor necrosis factor-alpha (TNF-α) and myeloperoxidase (MPO) activity. It also interrupted reactive oxygen species (ROS)-HMGB1loop as evident by restored liver reduced glutathione (GSH), elevated glutathione peroxidase (GPx) activity, along with decreased liver lipid peroxidation. Simultaneously, plumbagin significantly ameliorated apoptosis by amending the mRNA expressions of both anti-apoptotic (Bcl-2) and pro-apoptotic (Bax). The present results revealed that plumbagin is endowed with hepatoprotective activity ascribed to its antioxidant, anti-inflammatory and anti-apoptotic properties which are partially mediated through dampening of HMGB1 expression.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Biomarkers; Cytoprotection; Disease Models, Animal; Enzymes; Glutathione; Glutathione Peroxidase; Hepatitis; HMGB1 Protein; Inflammation Mediators; Lipid Peroxidation; Liver; Male; Naphthoquinones; Oxidative Stress; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction

Breast cancer (BC) is the second leading cause of death among women in the US, and its subtype triple-negative BC (TNBC) is the most aggressive BC with poor prognosis. In the current study, we investigated the anticancer effects of the natural product plumbagin (PL) on racially different TNBC cells. The PL effects were examined in two TNBC cell lines: MDA-MB-231 (MM-231) and MDA-MB-468 (MM-468), representing Caucasian Americans and African Americans, respectively. The results obtained indicate that PL inhibited cell viability and cell proliferation and induced apoptosis in both cell lines. Notably, MM-468 cells were 5-fold more sensitive to PL than MM-231 cells were. Testing PL and Taxol® showed the superiority of PL over Taxol® as an antiproliferative agent in MM-468 cells. PL treatment resulted in an approximately 20-fold increase in caspase-3 activity with 3 μM PL in MM-468 cells compared with an approximately 3-fold activity increase in MM-231 cells with 8 μM PL. Moreover, the results indicate a higher sensitivity to PL in MM-468 cells than in MM-231 cells. The results also show that PL downregulated CCL2 cytokine expression in MM-468 cells by 30% compared to a 90% downregulation in MM-231 cells. The ELISA results confirmed the array data (35% vs. 75% downregulation in MM-468 and MM-231 cells, respectively). Moreover, PL significantly downregulated IL-6 and GM-CSF in the MM-231 cells. Indeed, PL repressed many NF-қB-regulated genes involved in the regulation of apoptosis, proliferation, invasion, and metastasis. The compound significantly downregulated the same genes (BIRC3, CCL2, TLR2, and TNF) in both types of cells. However, PL impacted five more genes in MM-231 cells, including BCL2A1, ICAM1, IKBKE, IL1β, and LTA. In conclusion, the data obtained in this study indicate that the quinone compound PL could be a novel cancer treatment for TNBC in African American women.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Black or African American; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chemokine CCL2; Female; Humans; Naphthoquinones; NF-kappa B; Paclitaxel; RNA, Messenger; Signal Transduction; Triple Negative Breast Neoplasms; Tumor Necrosis Factor-alpha; White People

β-Amyloid (Aβ) species probably exert neurotoxic effects in Alzheimer's disease. In the present study, the effect of antioxidant plumbagin was tested against Aβ25-35-induced neurotoxicity in the SH-SY5Y cell line. Cell viability was determined using an MTT assay. Antioxidant status was analyzed through antioxidant enzyme activities, nuclear factor erythroid 2-related factor 2 (Nrf-2), and its downstream protein expressions. Inflammatory response was determined through nuclear factor-κB (NF-κB) pathway and cytokine expressions. Aβ25-35 showed a decrease in cell viability in a concentration-dependent manner. The IC50 value was found to be 17 µM. Pretreatment with plumbagin prevented Aβ25-35-induced toxicity by improving the cell viability up to 96%. Plumbagin inhibited Aβ25-35-induced oxidative stress by decreasing reactive oxygen species and lipid peroxidation. Aβ25-35-induced redox imbalance caused decreased Nrf-2 expression, with downregulation Nrf-2 target proteins heme oxygenase 1 and NAD(P)H dehydrogenase (quinone 1) during Aβ25-35 treatment. However, plumbagin improved the antioxidant defense system by increasing Nrf-2 expression with concomitant upregulation in heme oxygenase 1 and NAD(P)H dehydrogenase (quinone 1). Aβ25-35 induced inflammatory response through upregulated NF-κB, cyclooxygenase-2, and inducible nitric oxide synthase levels. Plumbagin exerted anti-inflammatory effects by decreasing NF-κB, cyclooxygenase-2, and inducible nitric oxide synthase levels. Aβ25-35-induced increases in proinflammatory cytokine (interleukin-8, interleukin-6, and monocyte chemoattractant protein-1) expressions were suppressed by plumbagin pretreatment. Altogether, the present study indicates that plumbagin prevents redox status and inflammatory activation during Aβ25-35-induced toxicity by modulating the antioxidant defense system and Nrf-2 signaling.

Topics: Antioxidants; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Humans; Naphthoquinones; Neuroprotective Agents; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Up-Regulation

Plumbagin (PLB) is the primary component of the traditional Chinese medicine Baihua Dan, and possesses anti-infection and anticancer effects, with the ability to enhance the sensitivity of tumor cells to radiation therapy. However, the anticancer effect of PLB on nasopharyngeal carcinoma and the underlying mechanisms remain unclear. In this study, we investigated the anticancer effects of PLB on nasopharyngeal carcinoma 6-10B cells and clarified its molecular mechanisms in vitro. The results showed that PLB was effective against 6-10B cells proliferation in a dose-dependent manner by inducing G2/M phase cell cycle arrest. Furthermore, our data showed that PLB induced reactive oxygen species accumulation, which inhibited the GSK3β/STAT3 pathway and arrested the G2/M phase. Therefore, our results provided new insight into the mechanism of the antitumor effects of PLB, supporting PLB as a prospective therapeutic drug in nasopharyngeal carcinoma by modulating intracellular redox balance.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; G2 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; Humans; M Phase Cell Cycle Checkpoints; Naphthoquinones; Nasopharyngeal Carcinoma; Oxidation-Reduction; Reactive Oxygen Species; STAT3 Transcription Factor; Up-Regulation

Plumbagin, a medicinal plant-derived 5-hydroxy-2-methyl-1,4-naphthoquinone, is an emerging drug with a variety of pharmacological effects, including potent anticancer activity. We have previously shown that plumbagin improves the efficacy of androgen deprivation therapy (ADT) in prostate cancer and it is now being evaluated in phase I clinical trial. However, the development of formulation of plumbagin as a compound with sparing solubility in water is challenging.. We have formulated plumbagin-loaded nanoemulsion using pneumatically controlled high pressure homogenization of oleic acid dispersions with polyoxyethylene (20) sorbitan monooleate as surfactant. Nanoemulsion formulations were characterized for particle size distribution by dynamic light scattering (DLS). The kinetics of. Particle size distribution of nanoemulsions is tunable and depends on the surfactant concentration. Nanoemulsion formulations of plumbagin with 1-3.5% (w/w) of surfactant showed robust stability of size distribution over time. Plumbagin-loaded nanoemulsion with average hydrodynamic diameter of 135 nm showed exponential release of plumbagin with a half-life of 6.1 h in simulated gastric fluid, 7.0 h in simulated intestinal fluid, and displayed enhanced antiproliferative effect toward prostate cancer cells PTEN-P2 compared to free plumbagin.. High drug-loading capacity, retention of nanoparticle size, kinetics of release under simulated physiological conditions, and increased antiproliferative activity indicate that oleic-acid based nanoemulsion formulation is a suitable delivery system of plumbagin.

Topics: Animals; Cell Death; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Drug Liberation; Emulsions; Male; Mice; Nanoparticles; Naphthoquinones; Particle Size; Prostatic Neoplasms; Surface-Active Agents

Lung cancer is the second most commonly diagnosed cancer and the leading cause of cancer deaths in both men and women in the United States. It has been recently demonstrated that osteopontin (OPN) effectively inhibits cofilin activity through the focal adhesion kinase (FAK)/AKT/Rho-associated kinase (ROCK) pathway to induce the invasion of human non-small cell lung cancer (NSCLC) cells. Plumbagin was isolated from the roots of the medicinal plant Plumbago zeylanica L. and has been reported to possess anticancer activities. However, the molecular mechanisms by which plumbagin inhibits the invasion of cancer cells is still unclear. In this study, the anti-invasive and anti-metastatic mechanisms of plumbagin were investigated in OPN-treated NSCLC A549 cells. OPN effectively induced the motility and invasion of NSCLC A549 cells and H1299 cells, which was strongly suppressed by plumbagin with no evidence of cytotoxicity. In addition, lamellipodia formation at the leading edge of cells by OPN was dramatically decreased in plumbagin-treated cells. Plumbagin caused an effective inhibition in OPN-induced the expression of ROCK1 as well as the phosphorylation of LIM kinase 1 and 2 (LIMK1/2), and cofilin. OPN-induced the phosphorylation of FAK and AKT was impaired without affecting their total forms by plumbagin treatment. OPN facilitated metastatic lung colonization, which was effectively suppressed in plumbagin-treated mice. Taken together, these results suggest that plumbagin reduces OPN-induced the invasion of NSCLC A549 cells, which resulted from inhibiting the ROCK pathway mediated by the FAK/AKT pathway and suppresses lung metastasis in vivo.

Topics: A549 Cells; Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Cell Movement; Humans; Lung; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Neoplasm Invasiveness; Osteopontin; Plumbaginaceae; Protein Kinase Inhibitors; rho-Associated Kinases; Signal Transduction

This study was designed to assess the effects of plumbagin on isoflurane-induced neurotoxicity.. Neonatal Sprague Dawley rat pups were treated with plumbagin (50, 100 or 150 mg/kg body weight, orally) from postnatal day 2. The pups on postnatal day 7 were subjected to isoflurane (0.75%) exposure for 6 h. Neuronal apoptosis in the hippocampal tissues was detected by TUNEL assay and FluroJade B staining following isoflurane exposure. Protein expressions were analysed by immunoblotting. RT-PCR was performed to assess mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB.. We observed reduced apoptosis in hippocampal CA1, CA3 and dentate gyrus regions along with severely reduced pro-apoptotic factors (Bad, Bax and cleaved caspase-3) expression and raised levels of Bcl-2, Bcl-xL, survivin, xIAP and cIAPs (cell survival proteins) in plumbagin supplemented rats. Decrease in the levels of JNK, phospho-JNK, c-Jun and phospho-c-Jun with enhanced ERK1/2 levels was observed on plumbagin pretreatment. Down-regulated PI3K/Akt signalling following isoflurane was activated by plumbagin as evidenced by raised PI3K/Akt pathway proteins - mTORc1, Akt, phospho-Akt, GSK-3β, phospho-GSK-3β, PTEN and NF-κBp65 in the hippocampal tissues as detected by Western blotting. The mRNA levels were enhanced on plumbagin supplementation.. Plumbagin exerted its neuroprotective effects by effectively suppressing isoflurane-induced neuronal apoptosis via regulating BDNF-TrkB-PI3/Akt and ERK/JNK signalling.

Topics: Anesthetics, Inhalation; Animals; Animals, Newborn; Apoptosis; Brain-Derived Neurotrophic Factor; Dose-Response Relationship, Drug; Hippocampus; In Situ Nick-End Labeling; Isoflurane; MAP Kinase Signaling System; Naphthoquinones; Neuroprotective Agents; Neurotoxicity Syndromes; Phosphatidylinositol 3-Kinases; Rats; Rats, Sprague-Dawley; Receptor, trkB; Signal Transduction

Although the patients with t(8;21) acute myeloid leukemia (AML) have a favorable prognosis compared with other non-acute promyelocytic leukemia AML patients, only ~50% patients with this relatively favorable subtype can survive for 5 years and refractory/relapse is common in clinical practice. So it is necessary to find novel agents to treat this type of AML. In this study, the effects and the mechanisms of plumbagin and recombinant soluble tumor necrosis factor‑α-related apoptosis-inducing ligand (rsTRAIL) on leukemic Kasumi‑1 cells were primarily investigated. Plumbagin and/or rsTRAIL could significantly inhibit the growth of Kasumi‑1 cells and induce apoptosis in vitro and in vivo. Plumbagin enhanced TRAIL-induced apoptosis of Kasumi‑1 cells in association with mitochondria damage, caspase activation, upregulation of death receptors (DRs) and decreased cFLIP expression. The effects of plumbagin on the expression of DR5, Bax and cFLIP could be partially abolished by the reactive oxygen species (ROS) scavenger NAC. Glutathione (GSH) depletion by plumbagin increased the production of ROS. In vivo, there was no obvious toxic pathologic change in the heart, liver and kidney tissues in any of the groups. Comparing with the control mice, a significantly increased number of apoptotic cells were observed in the combined treated mice by flow cytometry. Plumbagin also increased the expression of DR4 and DR5 in cells of xenograft tumors. Collectively, our results suggest that both plumbagin and rsTRAIL could be used as a single agent or synergistical agents to induce apoptosis of leukemic Kasumi‑1 cells in vitro and in vivo.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Leukemic; Glutathione; Humans; Leukemia, Myeloid, Acute; Mice; Mitochondria; Naphthoquinones; Reactive Oxygen Species; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Xenograft Model Antitumor Assays

Plumbagin is known to exhibit a broad range of biological activities including anti-cancer, antimicrobial and has been widely used traditionally. Nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) inhibitory and Nuclear factor (erythroid derived-2) like-2 (Nrf2) modulatory activities of Plumbagin have been reported already. In nerve injury model of neuropathy in rats, the role of NF-κB upregulation and declined antioxidant defence has been well recognized. So, we evaluated neuroprotective potential of Plumbagin in chronic constriction injury (CCI) of sciatic nerve induced neuropathic pain in male Sprague-Dawley rats.. Animals were tested for functional, behavioural and biochemical changes. Various markers associated with oxidative stress and inflammatory changes were assessed in the sciatic nerve and dorsal root ganglion (DRG) of the animals exposed to CCI mediated nerve injury.. CCI induced nerve injury led to long-lasting mechanical hyperalgesia, loss of hind limb function and abnormal pain sensation. Plumbagin treatment (10 and 20mg/kg, po) significantly and dose-dependently reversed mechanical hyperalgesia and other functional deficits. There was a marked increase in NF-κB and reduced Nrf2 levels in sciatic nerve and DRG following nerve injury. Plumbagin strengthened the antioxidant defence by improving Nrf2 levels and checked the neuroinflammation by decreasing NF-κB levels in sciatic nerve and DRG.. Together, these results suggested that Plumbagin alleviated CCI-induced neuropathic pain via antioxidant and anti-inflammatory mechanisms. Hence, the study suggests that Plumbagin may be useful for the management of trauma-induced neuropathic pain.

Topics: Adjuvants, Immunologic; Animals; Biomarkers; Hyperalgesia; Inflammation; Male; Naphthoquinones; Neuralgia; NF-E2-Related Factor 2; NF-kappa B; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy

Plumbagin is a natural naphthoquinone constituent isolated from the roots of medicinal plant Plumbago zeylanica L., and has demonstrated anti-proliferative and anti-invasion activities in various cancer cells. However, its effect on the migration and invasion of glioma cells has not been elucidated. Therefore, human glioma U87 and U251 cells were treated with plumbagin at 1.0 and 2.0 μM for 24 h, and cell migration and invasion were assessed with scratch wound healing and invasion assays. The results showed that plumbagin significantly inhibited the migration and invasion of U87 and U251 cells, suppressed the activity and expression of MMP-2/-9, and inhibited the nuclear translocation of transcription factors Sp1 in the U87 and U251 cells. Moreover, plumbagin reduced the level of p-PI3K and p-Akt in these cells. The combined treatment with plumbagin and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) reversed plumbagin-mediated inhibitory effects on MMP-2/-9 expression, cell migration and invasion. These findings suggest that the plumbagin-induced inhibition of glioma cell migration and invasion is closely associated with the downregulation of MMP-2/-9 expression and activity, and suppression of PI3K/Akt signaling pathway activation. Thus, plumbagin might be a potential anti-invasive agent in the treatment of glioma.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Glioma; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthoquinones; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Naphthoquinones; Nitric Oxide; Structure-Activity Relationship

The early detection and thus treatment of breast cancer bone metastasis remain a big challenge clinically. As the most abundant cells within bone tissue, osteocytes have been found to manipulate the activity of early cancer bone metastasis by its crosstalk with cancer cells and osteoclasts. However, conventional bone-targeting nanomedicine has limited bone-lesion specificity and ignores the vital role of osteocytes during breast cancer bone metastasis. Also, it lacks detailed insight into the therapeutic mechanisms, which hinders the following translational practice. Previously, we have shown that a combination of zoledronic acid (ZA) and plumbagin (PL) synergistically alleviates cancer-induced bone destruction. Herein, we further develop a pH-responsive bone-targeting drug delivery system, i.e., the ZA-anchored bimodal mesoporous slica covered gadolinium(III) upconversion nanoparticles loaded with PL, to detect and treat bone metastasis sensitively and specifically at an early stage. This multifunctional nanosystem can target osteocytes to release PL as controlled by pH, decreasing osteocytic RANKL expression synergistically through the structural simulation of adenosine phosphate, which competitively inhibits the phosphorylation of osteocytic protein kinase-a, cAMP-response element binding protein, extracellular regulated protein kinase, and c-Jun N-terminal kinase. More importantly, by establishing a breast cancer bone metastasis mice model via intracardiac injection, we show that tumoriogenesis and osteoclastogenesis can both be attenuated significantly. We thereby realize the effective theranostics of tiny bone metastasis in breast cancer bone metastasis. Our work highlights the significance of theranostic nanomedicine and osteocyte-targeting therapy in the treatment of early bone metastasis, which could be applied in achieving efficient theranostic effects for other bone diseases.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Delayed-Action Preparations; Drug Delivery Systems; Female; Gadolinium; Humans; Luminescent Agents; Mice; Mice, Nude; Nanoparticles; Naphthoquinones; Optical Imaging; Osteocytes; Silicon Dioxide; Theranostic Nanomedicine; Zoledronic Acid

Plumbagin inhibits the growth, metastasis, and invasion of prostate cancer (PCa). However, its lower bioavailability limits biopharmaceutical properties due to insolubility in water. Prostate-specific membrane antigen (PSMA) aptamer-targeted nanoparticles (NPs) significantly enhanced cytotoxicity in prostate epithelial cells. This study aimed to investigate the effects of plumbagin-loaded prostate-specific membrane antigen (PSMA) aptamer-targeted poly D,L-lactic-co-glycolic acid-b-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) on prostate cancer (PCa) in vitro.PLGA-PEG with a terminal carboxylic acid group (PLGA-PEG-COOH) was synthesized, and plumbagin was loaded on PLGA-PEG-COOH NPs using the nanoprecipitation method and characterized by field emission scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser light scattering. The uptake and distribution of plumbagin-NPs in human PCa LNCaP cells were investigated by fluorescent labeling. Subsequently, PSMA antibody-targeted PLGA-PEG-COOH NPs (targeted NPs) were prepared by covalent binding and characterized by x-ray photoelectron spectroscopy. Furthermore, the anticancer activity of plumbagin-loaded, targeted NPs was compared with that of nontargeted NPs in LNCaP cells in vitro.Plumbagin-NPs (diameter of 189.4 ± 30.6 nm and zeta potential of -17.1 ± 3.7 mV) were optimized based on theoretical drug loading of 5% and a ratio of water:acetone of 3:1. During the first 2 hours, the cumulative release rate of the drug was 66.4 ± 8.56%. Moreover, plumbagin-targeted NPs with nitrogen atoms were prepared. The uptake rate was 90% at 0.5 hours for targeted and nontargeted NPs. The IC50 of targeted NPs and nontargeted NPs was 32.59 ± 8.03 μM and 39.02 ± 7.64 μM, respectively.Plumbagin-loaded PSMA aptamer-targeted NPs can be used in targeted chemotherapy against PCa.

Topics: Antigens, Surface; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Delayed-Action Preparations; Dose-Response Relationship, Drug; Drug Carriers; Drug Evaluation, Preclinical; Drug Liberation; Glutamate Carboxypeptidase II; Humans; Male; Nanoparticles; Naphthoquinones; Particle Size; Prostatic Neoplasms

Critical roles for liver sinusoidal endothelial cells (LSECs) in liver fibrosis have been demonstrated, while little is known regarding the underlying molecular mechanisms of drugs delivered to the LSECs. Our previous study revealed that plumbagin plays an antifibrotic role in liver fibrosis. In this study, we investigated whether plumbagin alleviates capillarization of hepatic sinusoids by downregulating endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), laminin (LN), and type IV collagen on leptin-stimulated LSECs. We found that normal LSECs had mostly open fenestrae and no organized basement membrane. Leptin-stimulated LSECs showed the formation of a continuous basement membrane with few open fenestrae, which were the features of capillarization. Expression of ET-1, VEGF, LN, and type IV collagen was enhanced in leptin-stimulated LSECs. Plumbagin was used to treat leptin-stimulated LSECs. The sizes and numbers of open fenestrae were markedly decreased, and no basement membrane production was found after plumbagin administration. Plumbagin decreased the levels of ET-1, VEGF, LN, and type IV collagen in leptin-stimulated LSECs. Plumbagin promoted downregulation of ET-1, VEGF, LN, and type IV collagen mRNA. Altogether, our data reveal that plumbagin reverses capillarization of hepatic sinusoids by downregulation of ET-1, VEGF, LN, and type IV collagen.

Topics: Animals; Basement Membrane; Capillaries; Collagen Type IV; Down-Regulation; Endothelial Cells; Endothelin-1; Hepatic Veins; Hepatocytes; Laminin; Liver; Liver Cirrhosis; Male; Naphthoquinones; Rats; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A

Osteosarcoma is the most common primary bone tumor that occurs in children and adolescents. Osteosarcoma has a poor prognosis and is often unresponsive to chemotherapy. Therefore, it remains a challenge to identify a novel strategy to effectively treat osteosarcoma. The present study demonstrated a novel opportunity in osteosarcoma treatment using the natural compound plumbagin. Plumbagin reduced cell viability in osteosarcoma cells but not normal bone cells, as determined by MTT assay and colony formation assay. Plumbagin induced cell apoptosis by mitochondrial dysfunction, which in turn promoted Ca2+ release and endoplasmic reticulum (ER)‑stress, as determined by DAPI staining assay, DNA fragmentation assay, flow cytometry and western blotting analysis. In addition, plumbagin improved reactive oxygen species (ROS) generation, as determined by flow cytometry. Finally, these apoptotic cascades activated caspase‑3 and caspase‑9 to elicit apoptosis response. Our results demonstrated the anticancer effect of plumbagin by inducing cell apoptosis in osteosarcoma cells. In conclusion, plumbagin activated the apoptosis signaling pathway through eliciting ROS, ER stress, mitochondria dysfunction, and finally causing caspase activation. These results indicated that plumbagin may serve as potential antitumor drug by its multifunctional effects in osteosarcoma.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bone Neoplasms; Calcium; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Stress; Humans; Mitochondria; Models, Biological; Naphthoquinones; Osteosarcoma; Reactive Oxygen Species; Signal Transduction

Topics: Animals; Anti-Inflammatory Agents; Cell Line, Transformed; Cytokines; Dose-Response Relationship, Drug; Gene Expression Regulation; Lipopolysaccharides; Lysine; Mice; Microglia; Naphthoquinones; Nitric Oxide; Nitric Oxide Synthase Type II

Plumbagin is a candidate drug for the treatment of prostate cancer. Previous observations indicated that it may improve the efficacy of androgen deprivation therapy (ADT). This study evaluates the effectiveness of treatment with combinations of plumbagin and alternative strategies for ADT in mouse models of prostate cancer to support its clinical use.. Plumbagin was administered per oral in a new sesame oil formulation. Standard toxicology studies were performed in rats. For tumor growth studies, mouse prostate cancer cell spheroids were placed on top of grafted prostate tissue in a dorsal chamber and allowed to form tumors. Mice were separated in various treatment groups and tumor size was measured over time by intra-vital microscopy. Survival studies were done in mice after injection of prostate cancer cells in the prostate of male animals. Androgen receptor (AR) levels were analyzed by Western blot from prostate cancer cells treated with plumbagin.. Plumbagin caused a decrease in AR levels in vitro. In mice, plumbagin at 1 mg/kg in sesame oil displayed low toxicity and caused a 50% tumor regression when combined with castration. The combination of plumbagin with various forms of chemical ADT including treatment with a GnRH receptor agonist, a GnRH receptor antagonist, or CYP17A1 inhibitors, outperformed ADT alone, increasing mouse survival compared to the standard regimen of castration alone. In contrast, the combination of plumbagin with AR antagonists, such as bicalutamide and enzalutamide, showed no improvement over AR antagonists alone. Thus, plumbagin is effective in combination with drugs that prevent the synthesis of testosterone or its conversion to dihydrotestosterone, but not with drugs that bind to AR.. Plumbagin significantly improves the effect of ADT drugs currently used in the clinic, with few side effects in mice.

Topics: Androgen Receptor Antagonists; Animals; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Female; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Naphthoquinones; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Toxicity Tests; Treatment Outcome; Tumor Burden

The naphthoquinone droserone (

Topics: Animals; Antiviral Agents; Cell Survival; Cells, Cultured; Chlorocebus aethiops; Dioncophyllaceae; In Vitro Techniques; Inhibitory Concentration 50; Magnoliopsida; Measles; Measles virus; Naphthoquinones; Vero Cells

Melanoma is a highly drug resistant cancer. To circumvent this problem, a class of synergistically acting drug combinations, which inhibit multiple key pathways in melanoma cells, could be used as one approach for long-term treatment of this deadly disease. A screen has been undertaken on cell lines to identify those that could be combined to synergistically kill melanoma cells. Plumbagin and Celecoxib are two agents that were identified to synergistically kill melanoma cells by inhibiting the COX-2 and STAT3 pathways, which are constitutively activated in up to 70% of melanomas. The combination of these two drugs was more effective at killing melanoma cells than normal cells and decreased cellular proliferation as well as induced apoptosis of cultured cells. The drug combination inhibited development of xenograft melanoma tumors by up to 63% without affecting animal weight or blood biomarkers of organ function, suggesting negligible toxicity. Mechanistically, combination of Celecoxib and Plumbagin decreased melanoma cell proliferation and retarded vascular development of tumors mediated by inhibition of COX-2 and STAT3 leading to decreased levels of key cyclins key on which melanoma cell were dependent for survival.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Celecoxib; Cell Line, Tumor; Cell Proliferation; Cyclins; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; G2 Phase Cell Cycle Checkpoints; Humans; Melanoma; Mice, Nude; Naphthoquinones; Signal Transduction; Skin Neoplasms; STAT3 Transcription Factor; Time Factors; Tumor Burden; Xenograft Model Antitumor Assays

Using multiple drugs to kill cancer cells can decrease drug resistance development. However, this approach is frequently limited by the bioavailability and toxicity of the combined agents and delivery at ratios to specific locations that synergistically kill cancer cells. Loading the individual agents into a nanoparticle that releases the drugs at synergizing ratios at a single location is one approach to resolve this concern. Celecoxib and plumbagin are two drugs that were identified from a screen to synergistically kill melanoma cells compared with normal cells. Combined use of these agents by traditional approaches was not possible due to poor bioavailability and toxicologic concerns. This study details the development of a nanoliposomal-based agent containing celecoxib and plumbagin, called CelePlum-777, which is stable and releases these drugs at an optimal ratio for maximal synergistic killing efficacy. CelePlum-777 was more effective at killing melanoma than normal cells and inhibited xenograft melanoma tumor growth by up to 72% without apparent toxicity. Mechanistically, the drug combination in CelePlum-777 led to enhanced inhibition of melanoma cell proliferation mediated by decreasing levels of key cyclins important for cancer cell proliferation and survival, which was not observed with the individual agents. Thus, a novel nanoparticle-based drug has been developed containing celecoxib and plumbagin that lacks toxicity and delivers the agents at a synergistically killing drug ratio to kill cancer cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Celecoxib; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Drug Compounding; Drug Stability; Drug Synergism; Female; Humans; Liposomes; Melanoma; Mice; Nanoparticles; Naphthoquinones; STAT3 Transcription Factor; Tumor Burden; Xenograft Model Antitumor Assays

Tumor occurrence and development are very complicated processes. In addition to the roles of exogenous carcinogenic factors, the body's internal factors also play important roles. These factors include the host response to the tumor and the tumor effect on the host. In particular, the proliferation, migration and activation of endothelial cells are involved in tumor angiogenesis. Angiogenesis is one of the hallmarks of cancer. In this study, we investigate whether plumbagin can abrogate angiogenesis-mediated tumor growth in hepatocellular carcinoma (HCC) and, if so, through which molecular mechanisms. We observed that in co-cultures of the human endothelial cell line EA.hy926 and the human hepatoma cell line SMMC-7721 and Hep3B, the hepatoma cells induced migration, invasion, tube formation and viability of the EA.hy926 cells in vitro, and these processes were inhibited by plumbagin. Real-Time PCR, Western Blot and Immunofluorescence staining showed that plumbagin treatment suppressed expression of angiogenesis pathways (PI3K-Akt, VEGF/KDR and Angiopoietins/Tie2) and angiogenic factors (VEGF, CTGF, ET-1, bFGF),which is associated with tumor angiogenesis in cancer cells and xenograft tumor tissues. Furthermore, plumbagin was also found to significantly reduce tumor growth in an orthotopic HCC mouse model and to inhibit tumor-induced angiogenesis in HCC patient xenografts. Taken together, our findings strongly suggest that plumbagin might be a promising anti-angiogenic drug with significant antitumor activity in HCC.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Endothelial Cells; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Neoplasm Invasiveness; Neovascularization, Pathologic; Signal Transduction; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Recent studies have shown that plumbagin has anti-inflammatory, anti-allergic, antibacterial, and anti-cancer activities; however, it has not yet been shown whether plumbagin suppresses alpha-melanocyte stimulating hormone (α-MSH)-induced melanin synthesis to prevent hyperpigmentation. In this study, we demonstrated that plumbagin significantly suppresses α-MSH-stimulated melanin synthesis in B16F10 mouse melanoma cells. To understand the inhibitory mechanism of plumbagin on melanin synthesis, we performed cellular or cell-free tyrosinase activity assays and analyzed melanogenesis-related gene expression. We demonstrated that plumbagin directly suppresses tyrosinase activity independent of the transcriptional machinery associated with melanogenesis, which includes micropthalmia-associated transcription factor (

Topics: alpha-MSH; Animals; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Keratinocytes; Melanins; Melanoma, Experimental; Mice; Monophenol Monooxygenase; Naphthoquinones; Pigmentation; Signal Transduction

Inflammation and inflammatory cytokines have been reported to play vital roles in the development of osteoarthritis (OA). Plumbagin, a quinonoid compound extracted from the roots of medicinal herbs of the Plumbago genus, has been reported to have anti-inflammatory effects. However, the anti-inflammatory effects of plumbagin on OA have not been reported. This study aimed to assess the effects of plumbagin on human OA chondrocytes and in a mouse model of OA induced by destabilization of the medial meniscus (DMM). In vitro, human OA chondrocytes were pretreated with plumbagin (2, 5, 10 μM) for 2 h and subsequently stimulated with IL-1β for 24 h. Production of NO, PGE2, MMP-1, MMP-3, and MMP-13 was evaluated by the Griess reagent and ELISAs. The messenger RNA (mRNA) expression of COX-2, iNOS, MMP-1, MMP-3, MMP-13, aggrecan, and collagen-II was measured by real-time PCR. The protein expression of COX-2, iNOS, p65, p-p65, IκBα, and p-IκBα was detected by Western blot. The protein expression of collagen-II was evaluated by immunofluorescence. In vivo, the severity of OA was determined by histological analysis. We found that plumbagin significantly inhibited the IL-1β-induced production of NO and PGE2; expression of COX-2, iNOS, MMP-1, MMP-3, and MMP-13; and degradation of aggrecan and collagen-II. Furthermore, plumbagin dramatically suppressed IL-1β-stimulated NF-κB activation. In vivo, treatment of plumbagin not only prevented the destruction of cartilage and the thickening of subchondral bone but also relieved synovitis in mice OA models. Taken together, these results suggest that plumbagin may be a potential agent in the treatment of OA.

Topics: Adjuvants, Immunologic; Animals; Chondrocytes; Disease Progression; Humans; Inflammation; Interleukin-1beta; Mice; Naphthoquinones; NF-kappa B; Osteoarthritis; Signal Transduction

Context • Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer-related deaths in the world. The current treatments include surgery and chemotherapy, either alone or in combination with radiotherapy, but the prognosis for patients with GC is usually poor. A safe and effective chemopreventive treatment for this malignant disease is urgently needed. Objective • The study intended to investigate the effects and underlying mechanisms of plumbagin, a quinonoid constituent that is derived from the roots of the medicinal plant Plumbago zeylanica, which exhibits potent anticancer properties against a number of cancers. Design • The in vitro study used the human GC cell line SGC-7901. Setting • All experiments were conducted at the Hubei University of Chinese Medicine and Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Intervention • SGC-7901 cells were cultured in 30-mm dishes and treated with plumbagin at concentrations of 0, 5, 10, to 20 μmol/L. The cells were incubated with 10 μmol/L plumbagin for different amounts of time (0, 2, 4, 8, 12, and 24 h) in contact with the cancer cells. Outcome Measures • The cell viability was examined using a cell counting kit-8 viability assay, and the cell proliferation rate was determined using a 5-ethynyl-2'-deoxyuridine incorporation assay. The cell cycle distribution was assessed by flow cytometry using propidium iodide staining, and Western blotting was used to assess the expression of BAX, BCL-2, and caspase-3 and to identify any downregulation in the activation of transcription 3 (STAT3), protein kinase B (Akt), and extracellular signal-regulated kinase (ERK1/2). Results • The plumbagin concentrations of 5-20 mmol/L reduced the viability of the GC cells in a dependent manner. Plumbagin suppressed the expression of BAX, BCL-2, pro-caspase-3, and cleaved-caspase-3. It also restrained the expression and phosphorylation of STAT3 and decreased the phosphorylation of Akt1 but did not change the total protein or phosphorylation levels of ERK1/2. Conclusions • Plumbagin inhibits cell apoptosis in human GC cells, and that effect may be related with its ability to suppress phosphorylation of STAT3 and Akt. Given those 2 effects, plumbagin may be a promising agent in the treatment of gastric cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Humans; Naphthoquinones; Phytotherapy; Plumbaginaceae; Signal Transduction; STAT3 Transcription Factor; Stomach Neoplasms

Plumbagin (PLB), a natural naphthoquinone from the traditional folk medicines Plumbago zeylanica, Dionaea muscipula, or Nepenthes gracilis, has been documented possessing a wide variety of pharmacological activities. Although PLB demonstrates anticancer activity in multiple types of malignant cells, the cellular targets of PLB have not been well defined and remained only partially understood. We reported here that PLB selectively inhibits TrxR and elicits reactive oxygen species in human promyelocytic leukemia HL-60 cells, which leads to elevation of GSSG/GSH ratio and decrease of cellular thiol pool. As a consequence, PLB disturbs the cellular redox homeostasis, induces oxidative stress-mediated apoptosis and eventually selectively kills HL-60 cells. Inhibition of TrxR by PLB thus discloses an unprecedented mechanism underlying the anticancer efficacy of PLB, and sheds light in considering the usage of PLB as a promising cancer therapeutic agent.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Survival; HEK293 Cells; HeLa Cells; Hep G2 Cells; HL-60 Cells; Humans; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Protein Binding; Reactive Oxygen Species; Thioredoxin-Disulfide Reductase

Osteoarthritis (OA) is an inflammatory disorder dealing with the focal degradation of articular cartilage. Oxidative stress and inflammation are the major events in OA. The present study aimed at identifying the mechanism of the potent antioxidant, plumbagin, in protecting against hydrogen peroxide (H2O2)‑induced chondrocyte oxidative stress and inflammatory signaling. Oxidative stress was determined by measuring reactive oxygen species, lipid peroxidation, non‑enzymic (glutathione; GSH) and enzymic antioxidant activities (GSH, glutathione S‑transferase, glutathione peroxidase, superoxide dismutase, catalase). Expression levels of nuclear factor (erythroid‑derived 2)‑like 2 (Nrf‑2), heme oxygenase 1 (HO‑1), NAD(P)H:quinone oxidoreductase 1 (NQO‑1), nuclear factor‑κB (NF‑κB), cyclooxygenase‑2 (COX‑2) and inducible NO synthase (iNOS) were determined by western blot analysis. Pro‑inflammatory cytokine expression levels were assessed using ELISA. Results from reactive oxygen species generation, lipid peroxidation content and antioxidant enzyme activities demonstrated that plumbagin significantly inhibited oxidative stress status in H2O2‑induced chondrocytes. In addition, plumbagin modulated transcription factors involved in redox and inflammation regulation, including NF‑κB and Nrf‑2, by nuclear expression. plumbagin enhanced antioxidant status by increasing the expression levels of Nrf‑2 target genes, including HO‑1 and NQO‑1. An anti‑inflammatory effect against chondrocyte‑induced inflammation was demonstrated by downregulating COX‑2, iNOS and pro‑inflammatory cytokine expression levels (tumor necrosis factor‑α, interleukin (IL)‑6 and IL‑8). The present study identified strong evidence for a protective role of plumbagin against H2O2-induced oxidative stress and inflammation in chondrocytes by modulating redox signaling transcription factors.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cell Survival; Cells, Cultured; Chondrocytes; Humans; Hydrogen Peroxide; Naphthoquinones; NF-E2-Related Factor 2; NF-kappa B; Osteoarthritis; Oxidative Stress; Rats

The glucose regulated protein 78 (GRP78) is a major chaperone of the endoplasmic reticulum, and a prosurvival component of the unfolded protein response. GRP78 is upregulated in many types of cancers, including breast cancer. Research has suggested that GRP78 overexpression confers chemoresistance to anti-estrogen agents through a mechanism involving the inhibition of a pro-apoptotic BH3-only protein, Bik. In the present research the role of plumbagin, a naturally occurring naphthoquinone, in GRP78-associated cell death inhibition was examined. The results demonstrated that plumbagin inhibits GRP78 activity and GRP78 inhibition contributes to plumbagin-mediated cell death induction. Furthermore, Bik upregulation was associated with plumbagin-induced cell death and an increase in plumbagin-mediated Bik induction was observed upon GRP78 downregulation. Plumbagin sensitized estrogen-positive breast cancer cells to tamoxifen and the association of GRP78 inhibition and Bik upregulation in plumbagin-mediated cell sensitization was shown. Collectively, the results of this research suggest that plumbagin inhibits the antiapoptotic activity of GRP78 leading to Bik upregulation and apoptosis induction, which contributes to the sensitization of breast cancer cells to tamoxifen.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Chaperone BiP; Female; Heat-Shock Proteins; Humans; MCF-7 Cells; Membrane Proteins; Mitochondrial Proteins; Naphthoquinones; RNA Interference; Tamoxifen

Bioactive compounds, including phenols, flavonoids, and tannins, were quantified in leaves, stems and roots of methanol, n-butanol, diethyl ether and n- hexane extracts of Ceratostigina plumbaginoides Bunge. (Plumbaginaceae) ornamental plants. The antioxidant capacity was measured by the DPPH and linoleic acid assays. The total bioactive compounds, as well as the antioxidant capacities, were the highest in the leaves compared with stems and roots. The -methanolic, n-butanol, diethyl ether and n-hexane leaf extracts varied in their antibacterial and antifungal activities. In general, the most sensitive bacterium to leaf extracts was Bacillus cereus and the most resistant was Staphyllococcus aureus, while the most sensitive fungus was Aspergillusflavus and the most resistant one was Penicillium ochrochloron. As the methanolic leaf extract was the most active, it was subjected to column chromatography and two compounds were isolated and identified as 1 (5-hydroxy-2-methyl-1,4-naphthoquinone / plumbagin) and 2 (3,3'-biplumbagin). Compounds 1 and 2 showed the highest antibacterial and antifungal activities compared with other extracts tested. The MIC and MBC values for the most active compound 1 were in the range of 0.001 - 0.09 and 0.004 - 0.21 mg mL⁻¹, while MIC and MFC were determined as 0.001 - 0.11 and 0.002 - 0.19 mg mL⁻¹, respectively. The isolated compounds and leaf extracts showed also equal or higher antimicrobial activities compared with antibiotics/commercial reagents which indicate that the plant might be useful for drug development. This is the first report on the antibacterial and antifungal activities, as well as the antioxidant properties of the tested plant parts and isolated compounds.

Topics: Anti-Bacterial Agents; Anti-Infective Agents; Antifungal Agents; Antioxidants; Bacteria; Biphenyl Compounds; Fungi; Microbial Sensitivity Tests; Naphthoquinones; Picrates; Plant Extracts; Plant Leaves; Plumbaginaceae; Tannins

To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.. Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l(-1) and 10 mM L-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l(-1)). Treatments with 100 µM AgNO3 significantly increased intracellular plumbagin production by up to 7.6 mg g(-1) DW that was 4-fold more than the untreated root cultures (1.9 mg g(-1) DW). In contrast, treatments with 150 mg chitosan l(-1), 5 mM L-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g(-1) DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g(-1) DW, respectively.. Chitosan (150 mg l(-1)), L-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.

Topics: Cell Culture Techniques; Culture Media; Naphthoquinones; Phytochemicals; Plant Growth Regulators; Plant Roots; Plumbaginaceae

Plumbagin is the active compound isolated from plants used in traditional medicine for treatment of various diseases such as activities malaria, leishmaniasis, viral infections and cancers. The aim of the study was to investigate the permeability of plumbagin across Caco-2 (human epithelial colorectal adenocarcinoma) cell monolayer and its effects on the expression and function of P-glycoprotein. The integrity of Caco-2 cell monolayer was evaluated by measuring trans-epithelial electrical resistance and permeation (Papp) of Lucifer yellow across the cell monolayer. The effect of plumbagin on P-glycoprotein was detected by measuring its interference with the transport of the P-glycoprotein substrate (R123) and the effect on MDR-1 mRNA expression was detected by RT-PCR. The Papp of plumbagin (2-8 µM) for the apical to basolateral and basolateral to apical directions were 10.29-15.96 × 10(-6) and 7.40-9.02 × 10(-6) cm/s, respectively, with the efflux ratios of 0.57-0.73. Plumbagin is not either a substrate or inhibitor of P-glycoprotein. It did not interfere with the P-glycoprotein-mediated R123 transport across Caco-2 cell monolayer, as well as the function of P-glycoprotein and the expression of MDR-1 mRNA. Results suggest moderate permeability of plumbagin across the Caco-2 cell monolayer in both directions. The transport mechanism is likely to be a passive transport.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Caco-2 Cells; Cell Survival; Dose-Response Relationship, Drug; Electric Impedance; Humans; Intestinal Absorption; Isoquinolines; Naphthoquinones; Permeability; Rhodamine 123

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

Plumbagin is a derivative of napthoquinone which is isolated from the roots of plants in several families. These compound exhibits a wide range of biological and pharmacological activities including antimalarial, antibacterial, antifungal, and anticancer activities. The aim of the study was to investigate blood kinetics and tissue distribution of plumbagin in healthy and Plasmodium berghei-infected mice using Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) and radiochemical analysis by gamma counter. Plumbagin was labeled with (99m)technetium and the reducing agent stannous chloride dihydrate (50 μg/ml) at pH 6.5. Blood kinetics and tissue distribution of the radiolabeled plumbagin were investigated in healthy and P. berghei-infected mice (2 males and 2 females for each experimental group). In vitro and in vivo stability of plumbagin complex suggested satisfactory stability profiles of (99m)Tc-plumbagin complex in plasma and normal saline (92.21-95.47%) within 24 h. Significant difference in blood kinetics parameters (Cmax, AUC, t1/2, MRT, Vd, and CL) were observed between P. berghei-infected and healthy mice. The labeled complex distributed to all organs of both healthy and infected mice but with high intensity in liver, followed by lung, stomach, large intestine and kidney. Accumulation in spleen was markedly noticeable in the infected mice. Plumbagin-labeled complex was rapidly cleared from blood and major routes of excretion were hepatobiliary and pulmonary routes. In P. berghei-infected mice, t1/2 was significantly decreased, while Vd and CL were increased compared with healthy mice. Result suggests that malaria disease state influenced the pharmacokinetics and disposition of plumbagin. SPECT/CT imaging with radiolabeled (99m)Tc is a viable non-invasive technique that can be applied for investigation of kinetics and biodistribution of plumbagin in animal models.

Topics: Animals; Brain; Female; Gastric Mucosa; Humans; Hydrogen-Ion Concentration; Intestinal Mucosa; Kidney; Lung; Malaria; Male; Mice; Mice, Inbred ICR; Microsomes, Liver; Models, Animal; Myocardium; Naphthoquinones; Plasmodium berghei; Spleen; Technetium; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed

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

Eventhough the role of BRCA1/2 in hereditary prostatic cancer is being unleashed at a rapid rate; their optimal clinical management remains undefined. Cancer stem cells are thought to be responsible for cancer chemoresistance and relapse, thus they represent a significant concern for cancer prognosis and therapy. In this study, we have analyzed the effect of Plumbagin (PB) and structurally related naphthaquinones on BRCA1/2 silenced prostate cancer cells and the ability of PB to target stem cells. Our cell proliferation studies showed that both PC-3 and DU145 cells were more sensitive to PB, though all the compounds induced mitochondrial potential loss, DNA fragmentation and morphological changes which are indicative of apoptosis. Both BRCA1/2 siRNA transfected PC-3 and DU145 cells exhibited increased sensitivity to PB. Gene expression profiling post PB treatment in BRCA1/2 silenced cells revealed that PB has a putative role in tumor suppression in BRCA defective cancers. Using flow cytometric analysis we have proved that PB has the putative ability to directly target CSCs. Overall studies suggest that PB's antitumour mechanisms holds promise for novel therapeutic approaches against BRCA mutated cancers as well as CSCs.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Male; Naphthoquinones; Neoplastic Stem Cells; Prostate; Prostatic Neoplasms

The treatment of breast cancer-induced osteolysis remains a challenge in clinical settings. Here, we explored the effect and mechanism of combined treatment with zoledronic acid (ZA) and plumbagin (PL), a widely investigated component derived from Plumbago zeylanica, against breast cancer-induced osteoclastogenesis. We found that the combined treatment with PL and ZA suppressed cell viability of precursor osteoclasts and synergistically inhibited MDA-MB-231-induced osteoclast formation (combination index=0.28) with the abrogation of recombinant mouse receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of NF-κB/MAPK (nuclear factor-κB/mitogen-activated protein kinase) pathways. Molecular docking suggested a putative binding area within c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk) protease active sites through the structural mimicking of adenosine phosphate (ANP) by the spatial combination of PL with ZA. A homogeneous time-resolved fluorescence assay further illustrated the direct competitiveness of the dual drugs against ANP docking to phosphorylated JNK/Erk, contributing to the inhibited downstream expression of c-Jun/c-Fos/NFATc-1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1). Then, in vivo testing demonstrated that the combined administration of PL and ZA attenuated breast cancer growth in the bone microenvironment. Additionally, these molecules prevented the destruction of proximal tibia, with significant reduction of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclast cells and potentiation of apoptotic cancer cells, to a greater extent when combined than when the drugs were applied independently. Altogether, the combination treatment with PL and ZA could significantly and synergistically suppress osteoclastogenesis and inhibit tumorigenesis both in vitro and in vivo by simulating the spatial structure of ANP to inhibit competitively phosphorylation of c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk).

Topics: Adenine Nucleotides; Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Diphosphonates; Disease Models, Animal; Drug Synergism; Female; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Osteoclasts; Osteolysis; Phosphorylation; Random Allocation; Signal Transduction; Zoledronic Acid

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

The aim of the present study was to investigate the protective effect exerted by bone marrow mesenchymal stem cells (BMSCs) in combination with plumbagin on spinal cord injury (SCI) and explore the mechanism behind this protective effect. Firstly, BMSCs were extracted from male Sprague-Dawley rats, cultured in vitro, and identified by hematoxylin. Sprague-Dawley rats were then randomly divided into a control group, SCI model group, BMSC-treated group, a plumbagin-treated group, and a BMSC and plumbagin-treated group. After treatment with BMSCs combined with plumbagin, a Basso, Beattie and Bresnahan (BBB) test was carried out and the spinal cord water content was examined in order to analyze the effect of BMSCs combined with plumbagin on SCI. The myeloperoxidase (MPO), superoxide dismutase (SOD), malondialdehyde (MDA), nuclear factor-κB (NF-κB) p65 unit, tumor necrosis factor-α (TNF-α) levels were also detected. Moreover, nuclear factor erythroid 2‑related factor 2 (Nrf2), phosphoinositide 3-kinase (PI3K), phosphorylated (p-)Akt, p-p38 mitogen-activated protein kinase (MAPK), and p-extracellular-signal-regulated kinase (ERK) protein expression levels were measured using western blot analysis. Treatment with BMSCs combined with plumbagin significantly improved locomotor recovery and reduced the spinal cord water content after SCI. The increased MPO, MDA, NF-κB p65 and TNF-α levels were significantly suppressed and the decreased SOD was significantly increased in SCI rats. The suppression of Nrf2, p-Akt and p-ERK, as well as the promotion of p-p38 MAPK, were reversed by treatment with BMSCs combined with plumbagin. These effects suggest that treatment with BMSCs combined with plumbagin alleviates SCI through its effects on oxidative stress, inflammation, apoptotis and activation of the Nrf2 pathway.

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Inflammation; Locomotion; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Naphthoquinones; NF-E2-Related Factor 2; Oxidative Stress; Plumbaginaceae; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; Spinal Cord Injuries

Of the hematological disorders typified by poor prognoses and survival rates, T-cell acute lymphoblastic leukemia (T-ALL) is one of the most commonly diagnosed. Despite the development of new therapeutic agents, the treatment options for this cancer remain limited. In this manuscript, we investigated the anti-proliferative effects of plumbagin, mediated by the activation of mitogen-activated protein kinase (MAPK) pathways, and inhibition of NF-κB signaling; the human T-ALL MOLT-4 cell line was used as our experimental system. Plumbagin is a natural, plant derived compound, which exerts an anti-proliferative activity against many types of human cancer. Our experiments confirm that plumbagin induces a caspase-dependent apoptosis of MOLT-4 cells, with no significant cytotoxicity seen for normal peripheral blood mononuclear cells (PBMCs). Plumbagin also inhibited LPS-induced phosphorylation of p65, and the transcription of NF-κB target genes. Our results now show that plumbagin is a potent inhibitor of the NF-κB signaling pathway, and suppressor of T-ALL cell proliferation.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytokines; DNA Primers; Flow Cytometry; Humans; Immunosuppressive Agents; Leukocytes, Mononuclear; Lipopolysaccharides; Naphthoquinones; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Signal Transduction; Transcription, Genetic

Plumbagin, a naphthoquinone constituent of Plumbago zeylanica L. (Plumbaginaceae), is known to exhibit proapoptotic, antiangiogenic and antimetastatic effects in cancer cells. However, the effect of Plumbagin on breast cancer cells and the underlying molecular mechanism has not yet been elucidated.. MCF-7 (a human breast cancer cell line) was exposed different concentrations of Plumbagin (PG), and the anti-proliferative activity was evaluated by the MTT assay. The mechanism of action for the growth inhibitory activity of Plumbagin on MCF-7 cancer cells was evaluated using flow cytometry for cell cycle distribution, and western blot for assessment of expression of potential target proteins.. Plumbagin exhibited a significant anti-proliferative activity against human breast cancer cells. Flow cytometric analysis revealed that Plumbagin caused cell cycle arrest at G1 phase. The cell cycle arrest was well correlated with the inhibition of cyclin D1, cyclin E, and upregulation of tumor suppressor protein p53. It further inhibited the expression of anti-apoptotic Bcl-2 family members such as Bcl-xL and Bcl-2, and activated pro-apoptotic proteins like Bax and Bak.. These findings suggest that the anti-proliferative effect of Plumbagin is due to upregulation of p53 and p21 and suppression of G1 cell cycle regulators.

Topics: Antineoplastic Agents, Phytogenic; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53; Up-Regulation

BACKGROUND Plumbagin is a potent antioxidant with anti-inflammatory and anti-carcinogenic action. Myocardial ischemia/reperfusion injury results in organ damage through oxidative stress and inflammatory mechanisms. In this study, we analyzed the potential role of plumbagin against myocardial I/R injury in Wistar rats. MATERIAL AND METHODS Oxidative stress was measured through ROS, lipid peroxide content, and antioxidant enzyme activities. The expression of redox signaling and inflammatory proteins was analyzed through Western blotting. Inflammatory cytokine expressions were determined through ELISA. RESULTS Oxidative stress status was reduced by plumbagin by decreasing ROS and lipid peroxide levels in rats with myocardial I/R (MI/R) injury. Plumbagin regulated redox imbalance induced by I/R injury by modulating the transcription factors NF-κB and Nrf-2. Further, downstream targets of NF-κB (COX-2, iNOS) and Nrf-2 (HO-1, NQO1 and GST) expression were significantly downregulated by plumbagin treatment. Pro-inflammatory cytokine expressions were significantly abrogated by plumbagin treatment. CONCLUSIONS This study shows the protective role of plumbagin against myocardial I/R injury by regulating antioxidant and inflammatory mechanisms.

Topics: Animals; Anti-Inflammatory Agents; Anticarcinogenic Agents; Antioxidants; Apoptosis; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Myocardial Reperfusion Injury; Naphthoquinones; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Random Allocation; Rats; Rats, Wistar; Signal Transduction

Plumbagin, one of the constituents responsible for the various biological activities of Plumbago zeylanica has been demonstrated to possess antioxidant activity, which may inhibit lipid peroxidation in a dose- and time-dependent manner. In the present study, we aimed to examine the protective effects of plumbagin as well as the underlying mechansim through which plumbagin attenuates hydrogen peroxide (H2O2)-induced oxidative stress in nucleus pulposus cells (NPCs). For this purpose, the NPCs were incubated with fresh medium containing H2O2 (200 µM) at 37˚C in a humidified 5% CO2 atmosphere for 6 h, and cultured with various concentrations of plumbagin (0, 0.5, 1, 2, 5, 10 and 20 µM). Treatment with plumbagin significantly increased the viability of the H2O2-exposed NPCs in a dose‑dependent manner. Moreover, plumbagin significantly reduced the generation of reactive oxygen species, lipid peroxidation, as well as the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in the H2O2‑exposed NPCs. Glutathione (GSH) content, as well as the activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxdiase (GSH-Px) were increased. We found that the administration of plumbagin significantly inhibited the activity of caspase-9 and -3, and downregulated NF-κB expression and upregulated Nrf-2 expression in the H2O2-exposed NPCs. Taken together, these findings suggest that plumbagin exerts neuroprotective effects in NPCs by attenuating H2O2‑induced oxidative stress, inflammation and apoptosis through mediating the expression of NF-κB and Nrf-2.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Catalase; Chondrocytes; Dose-Response Relationship, Drug; Gene Expression Regulation; Glutathione; Glutathione Peroxidase; Hydrogen Peroxide; Inflammation; Male; Naphthoquinones; NF-E2-Related Factor 2; NF-kappa B; Nucleus Pulposus; Osteoblasts; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase

Plumbagin, (5-hydroxy-2-methyl-1,4-naphthoquinone), a natural substance found in the roots of plant species in the genus Plumbago, has been used as a traditional medicine against many diseases. In this study, Caenorhabditis elegans was used as a model for testing the anthelmintic effect of plumbagin. The compound exhibited a nematicidal effect against all stages of C. elegans: L4 was least susceptible, while L1 was most susceptible to plumbagin with an LC(50) of 220 and 156 μM, respectively. Plumbagin inhibited C. elegans development from L1 to adult stages with an IC(50) of 235 μM, and body length was also reduced at concentrations of 25 and 50 μg/ml. Brood sizes decreased from 203±6 to 43±6 and 18±3 eggs per hatch in plumbagin-treated worms at 10, 25, 50 μg/ml, respectively. Furthermore, plumbagin was lethal to strains resistant to the nematicides levamisole, albendazole, and ivermectin, indicating that it possesses a strong and unique nematicidal action. Plumbagin decreased the number of mitochondria in hypodermal and intestinal cells and body wall muscles and damaged the ultrastructure of these tissues. Taken together, plumbagin may be a new drug against parasitic nematodes.

Topics: Animals; Antinematodal Agents; Caenorhabditis elegans; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Naphthoquinones

Candida albicans and Staphylococcus aureus are opportunistic pathogens. Despite causing a number of independent infections, both pathogens can co-infect to cause urinary tract infections, skin infections, biofilm associated infections, sepsis and pneumonia. Infections of these two pathogens especially their biofilm associated infections are often difficult to treat using currently available anti-bacterial and anti-fungal agents. In order to identify a common anti-microbial agent which could confer a broad range of protection against their infections, we screened several phytochemicals and identified plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a phytochemical from Plumbago species as a potent antimicrobial agent against S. aureus and C. albicans, with a minimum inhibitory concentration of 5μg/ml. Antimicrobial activity of plumbagin was validated using an ex-vivo porcine skin model. For better understanding of the antimicrobial activity of plumbagin, a Drosophila melanogaster infection model was used, where D. melanogaster was infected using S. aureus and C. albicans, or with both organisms. The fly's survival rate was dramatically increased when infected flies were treated using plumbagin. Further, plumbagin was effective in preventing and dispersing catheter associated biofilms formed by these pathogens. The overall results of this work provides evidence that plumbagin, possesses an excellent antimicrobial activity which should be explored further for the treatment of S. aureus and C. albicans infections.

Topics: Animals; Anti-Infective Agents; Biofilms; Candida albicans; Candidiasis; Disease Models, Animal; Drosophila melanogaster; Female; Microbial Sensitivity Tests; Naphthoquinones; Phytochemicals; Plumbaginaceae; Staphylococcal Infections; Staphylococcus aureus; Survival Analysis; Treatment Outcome

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), the major active constituent of Plumbago indica L., has been shown to be effective against a wide range of infectious microbes. In this study, plumbagin has been evaluated in vitro for its antifungal combinatory effect with amphotericin B against Candida albicans (C. albicans) clinical isolates and anti-hepatitis C virus (HCV) activity. Antifungal activity was determined by broth microdilution method, and combinatory effect was evaluated by checkerboard assay according to ΣFIC indices, while cytotoxicity was determined by MTT assay. Anti-HCV activity was determined in infected Huh7.5 cells using quantitative real-time reverse transcription PCR, and cytotoxicity was evaluated by MTT assay. Plumbagin exerted inhibitory effect against all C. albicans strains with minimum inhibitory concentration values ranging from 7.41 to 11.24 µg/mL. The additive effect of plumbagin when combined with amphotericin B at concentrations of (0.12, 0.13 and 0.19, 1.81 µg/mL, respectively) was obtained against five of seven strains tested with ΣFIC ranging from 0.62 to 0.91. In addition, plumbagin was found to be used safely for topical application when combined with amphotericin B at concentrations corresponding to the additive effect. Plumbagin exerted anti-HCV activity compared with that of telaprevir with IC50 values of 0.57 and 0.01 μM/L, respectively, and selectivity indices SI = 53.7 and SI = 2127, respectively. Our results present plumbagin as a potential therapeutic agent in the treatment of C. albicans and HCV infections. Copyright © 2016 John Wiley & Sons, Ltd.

Topics: Amphotericin B; Antifungal Agents; Candida albicans; Hepacivirus; Humans; Naphthoquinones

We have earlier shown that Plumbagin (PB) can induce selective cytotoxicity to BRCA1 defective ovarian cancer cells; however, the effect of this molecule in BRCA1 mutated breast cancers has not been analyzed yet. Here, we report that reactive oxygen species (ROS) induced by PB resulted in DNA DSB and activates downstream signaling by ATR/ATM kinases and subsequent apoptosis. PB reduces DNA- dependent protein kinase (DNA-PK) expression and inhibits NHEJ (Non Homologous End Joining) activity in BRCA1 defective breast cancer cells. Also, PB induces apoptosis in two different BRCA1 conditional knock out murine models: MMTV-Cre; BRCA1(Co/Co) and WAP-Cre; BRCA1(Co/Co), at 2 mg/kg body weight, but 32 mg/kg of carboplatin (CN) was needed to induce apoptosis in them. This is the first study where two different tissue specific promoter driven transgenic mice models with BRCA1 exon 11 deletions are used for preclinical drug testing. The apoptosis induced by PB in HR (Homologous Recombination) defective triple negative BRCA1 mutant cell lines and in mouse models occur by inducing ROS mediated DNA DSB. The toxicity profile as compared with CN in transgenic mice provides evidence for PB's safer disposition as a therapeutic lead in breast cancer drug development.

Topics: Animals; Apoptosis; BRCA1 Protein; Cell Line, Tumor; DNA Breaks, Double-Stranded; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Naphthoquinones; Triple Negative Breast Neoplasms; Tumor Suppressor Proteins

Studies over the past decade and half have identified cancer stem cells (CSCs) to be responsible for tumorigenesis, invasion, sustenance of metastatic disease, radio- and chemo-resistance and tumor relapse. Recent reports have described the plasticity of breast CSCs (BCSCs) to shift between the epithelial and mesenchymal phenotypes via Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET) states as the reason for their invasive capabilities. Additionally, BRCA1 has been found to be a mammary stem cell fate determinant. However, it is not clear what would be the best marker that can be used for identifying CSCs in BRCA1 mutated cancers. Also, anticancer agents that can reduce CSC population in a BRCA1 defective condition have not been addressed so far.. Putative BCSCs were identified based on Hoechst exclusion, CD44(+)/24(-/low) expression and Aldehyde Dehydrogenase 1 (ALDH1) positivity using flow cytometry. The 'stemness' of the isolated ALDH1+ cells were analysed by immunofluorescence, western blotting for stem cell and EMT markers as well as in vitro mammosphere assays. Induction of Reactive Oxygen Species (ROS) by Plumbagin (PB) in BCSCs was assayed by Dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. Ovarian cancer xenografts treated with PB were subjected to immunohistochemical analysis to study the ability of PB to target CSCs.. We have confirmed that ALDH1 positivity is the best marker for the identification of BCSCs in BRCA1-defective breast cancer cell lines when compared to the CD marker profile and Side Population (SP) analysis. BRCA1 status was observed to be a determinant of the abundance of epithelial-like (ALDH1+) or mesenchymal-like (CD44(+)/24(-/low)) BCSCs, and the reconstitution of a full length, wild type BRCA1 in HCC1937 breast cancer cells possessing a mutated BRCA1, transforms them from 'stem-like' to more 'mesenchymal'. For the first time we have shown that Plumbagin (PB), a naturally occurring naphthoquinone which is predominantly a ROS inducer, could reduce BCSCs specifically in BRCA1-defective, basal-like cancer cells.. The best marker for identifying BCSCs in BRCA1 defective condition could be ALDH1 and that BRCA1 mutated BCSCs would be mostly 'stem like' than 'mesenchymal'. Also ROS inducers like PB could reduce BCSCs in BRCA1 defective cancers.

Topics: Animals; Antineoplastic Agents, Phytogenic; Biomarkers, Tumor; BRCA1 Protein; Breast Neoplasms; Carboplatin; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Humans; Inhibitory Concentration 50; Mice, SCID; Naphthoquinones; Neoplastic Stem Cells; Xenograft Model Antitumor Assays

Naphthoquinones are known to exhibit a broad range of biological activities against microbes, cancer and parasitic diseases and have been widely used in Indian traditional medicine. Plumbagin is a plant-derived naphthoquinone metabolite (5-hydroxy-2-methyl-1,4-naphthoquinone) reported to inhibit trypanothione reductase, the principal enzyme and a validated drug target involved in detoxification of oxidative stress in Leishmania. Here, we report the mechanistic aspects of cell death induced by plumbagin including physiological effects in the promastigote form and ultrastructural alterations in both promastigote and amastigote forms of Leishmania donovani which till now remained largely unknown. Our observations show that oxidative stress induced by plumbagin resulted in depolarization of the mitochondrial membrane, depletion in ATP levels, elevation of cytosolic calcium, increase in caspase 3/7-like protease activity and lipid peroxidation in promastigotes. Apoptosis-like cell death induction post plumbagin treatment was confirmed by biochemical assays like Annexin V/FITC staining, TUNEL as well as morphological and ultrastructural studies. These findings collectively highlight the mode of action and importance of oxidative stress inducing agents in effectively killing both forms of the Leishmania parasite and opens up the possibility of exploring plumbagin and its derivatives as promising candidates in the chemotherapy of Leishmaniasis.

Topics: Adenosine Triphosphate; Annexin A5; Apoptosis; Calcium; Caspases; Cell Death; Cells, Cultured; Cytosol; Fluorescein-5-isothiocyanate; In Situ Nick-End Labeling; Leishmania donovani; Lipid Peroxidation; Membrane Potential, Mitochondrial; Mitochondria; NADH, NADPH Oxidoreductases; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a natural naphthoquinone compound isolated from roots of Plumbago zeylanica L., has drawn a lot of attention for its plenty of pharmacological properties including antidiabetes and anti-cancer. The aim of this study was to investigate the effects of plumbagin on CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver and evaluate the potential herb-drug interactions using the cocktail approach. All CYP substrates and their metabolites were analyzed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plumbagin presented non-time-dependent inhibition of CYP activities in both human and rat liver. In humans, plumbagin was not only a mixed inhibitor of CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, but also a non-competitive inhibitor of CYP1A2, with Ki values no more than 2.16 μM. In rats, the mixed inhibition of CYP1A2 and CYP2D1, and competitive inhibition for CYP2B1, CYP2C11 and CYP2E1 with Ki values less than 9.93 μM were observed. In general, the relatively low Ki values of plumbagin in humans would have a high potential to cause the toxicity and drug interactions involving CYP enzymes.

Topics: Animals; Chromatography, Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drugs, Chinese Herbal; Humans; In Vitro Techniques; Kinetics; Male; Microsomes, Liver; Naphthoquinones; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry

Root cultures of the West African liana Triphyophyllum peltatum were initiated from stem explants of in vitro cultivated shoots. From these organ cultures, three new binaphthalenes, one binaphthoquinone, and two (bi)naphthalene glucosides were isolated, with substitution patterns related to those of the naphthylisoquinoline alkaloids, which are the "normal" main metabolites of T. peltatum. The structures of the diglucoside dioncoquinoside A (1) and of the axially chiral biaryls triphyoquinols A1 (3), A2 (4), and B (5), triphyoquinoside A (6), and triphyoquinone A (7) were elucidated by spectroscopic analysis (HRESIMS, 1D and 2D NMR) and by application of electronic circular dichroism (ECD) spectroscopy in combination with the exciton chirality method and quantum-chemical ECD calculations. The root cultures likewise produced the known alkaloids dioncophylline A (8), 5'-O-demethyldioncophylline A (9), dioncopeltine A (10), habropetaline A (11), and 5'-O-methyldioncophylline D (12a/b), the naphthalene glucoside plumbaside A (2), and the naphthoquinones plumbagin (13), droserone (14), and 8-hydroxydroserone (15).

Topics: Africa, Western; Alkaloids; Dioncophyllaceae; Isoquinolines; Molecular Structure; Naphthalenes; Naphthoquinones; Plant Extracts; Plant Roots

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, and mainly originates from an accumulation of abnormal B cells caused by the dysregulation of cell proliferation and apoptosis rates. The aberration of apoptosis-related genes in CLL cells results in defective apoptosis of CLL cells in response to traditional therapeutic medicine. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), a natural compound from Plumbago zeylinica, has been shown to exhibit pro-apoptotic activities in tumor cells. In the present study, we report that plumbagin effectively inhibited CLL cell viability with a lower dose compared to fludarabine, and inhibited cell proliferation in a dose-dependent manner. In addition, plumbagin promoted accumulation of MEC-1 cells in the S phase, and blocked cell cycle transition of HG3 cells from G0/G1 to S phase. Molecularly, plumbagin markedly induced CLL cell apoptosis through reduction of Bcl-2, but through an increase in the Bax protein level. These results suggest that plumbagin may be considered as a potential anticancer agent for CLL therapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Proliferation; Cell Survival; Down-Regulation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Up-Regulation

Natural plant products are increasingly being used in cancer therapeutic studies due to their reduced normal cell toxicity. In this study, the anti-cancer properties of plumbagin, a naphthoquinone derivative extracted from the roots of Plumbago, were evaluated in breast cancer cells.. To evaluate the effects of plumbagin on breast cancer cell types, we employed a variety of techniques comprising cell viability, cell cycle assay, comet assay, western blotting, immunocytochemistry, measurement of telomerase activity, telomere restriction fragment length, quantitative fluorescence in situ hybridisation, along with gene expression analysis of untreated cells.. Plumbagin treatment induced cytotoxicity in human breast cancer cells along with cell cycle arrest, DNA damage and cell death leading to apoptosis. Plumbagin was also found to suppress the telomerase activity in cancer cells accompanied by telomere attrition. Telomere shortening was corroborated by reduced telomere fluorescence on chromosome ends and genome instability.. Together, these findings may suggest the application of plumbagin as adjuvant modality in breast cancer therapeutics.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Chromosome Aberrations; Clone Cells; DNA Breaks, Double-Stranded; DNA Damage; Female; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; In Situ Hybridization, Fluorescence; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Telomerase; Telomere; Telomere Homeostasis; Wound Healing

Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.

Topics: Acid Anhydride Hydrolases; Animals; Antitubercular Agents; Biofilms; Citric Acid Cycle; Clofazimine; Disease Models, Animal; Drug Resistance, Bacterial; Gene Expression; Glycerophosphates; Isoenzymes; Isoniazid; Meropenem; Mice; Mycobacterium tuberculosis; Naphthoquinones; Phosphotransferases (Phosphate Group Acceptor); Polyphosphates; Thienamycins; Tuberculosis Vaccines; Tuberculosis, Multidrug-Resistant; Vaccines, DNA; Xylose

Tamoxifen is widely used as the first line drug for estrogen receptor-positive subtype which is expressed in 70% of overall breast cancer patients. However, approximately 50% of these patients develop acquired resistance after 5 years of treatment, which is characterized by tumor recurrence and metastasis. The epithelial mesenchymal transition (EMT) is an important process in breast cancer invasion. Fundamentally, targeting the EMT represents a crucial therapeutic strategy for preventing or treating breast cancer metastasis. Plumbagin (PLB) is a natural naphthoquinone with significant anticancer effects against several types of tumor cells including breast cancer. In this study, we investigated the effect of PLB on human endocrine-resistant breast cancer cell growth, invasion and the possible mechanisms underlying such actions. PLB exhibited potent cytotoxic activity at a micromolar concentration against endocrine-resistant breast cancer cells. Interestingly, a fixed low concentration of PLB and tamoxifen combination resulted in an increase in growth inhibition in endocrine-resistant cells. In addition, PLB also significantly suppressed mesenchymal biomarker expressions that govern the EMT process, resulting in attenuated metastatic capabilities. In conclusion, PLB should be developed as a pharmacological agent for the use as a single treatment or in combination for endocrine-resistant breast cancer. Copyright © 2016 John Wiley & Sons, Ltd.

Topics: Anticoagulants; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; Naphthoquinones; Tamoxifen

The transcription factor c-Myb is essential for the proliferation of hematopoietic cells and has been implicated in the development of leukemia and other human cancers. Pharmacologic inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. By using a Myb reporter cell line, we have identified plumbagin and several naphthoquinones as potent low-molecular weight Myb inhibitors. We demonstrate that these compounds inhibit c-Myb by binding to the c-Myb transactivation domain and disrupting the cooperation of c-Myb with the coactivator p300, a major driver of Myb activity. Naphthoquinone-induced inhibition of c-Myb suppresses Myb target gene expression and induces the differentiation of the myeloid leukemia cell line HL60. We demonstrate that murine and human primary acute myeloid leukemia cells are more sensitive to naphthoquinone-induced inhibition of clonogenic proliferation than normal hematopoietic progenitor cells. Overall, our work demonstrates for the first time the potential of naphthoquinones as small-molecule Myb inhibitors that may have therapeutic potential for the treatment of leukemia and other tumors driven by deregulated Myb. Mol Cancer Ther; 15(12); 2905-15. ©2016 AACR.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; E1A-Associated p300 Protein; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Naphthoquinones; Protein Binding; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins c-myb; Reactive Oxygen Species

HER2-overexpressing breast cancers account for about 30% of breast cancer occurrences and have been correlated with increased tumor aggressiveness and invasiveness. The nuclear factor-κB (NF-κB) is overexpressed in a subset of HER2-positive breast cancers and its upregulation has been associated with the metastatic potential of HER2-overexpressing tumors. The present study aimed at determining the potential of plumbagin, a naturally occurring naphthoquinone, to inhibit the invasion of HER2-overexpressing breast cancer cells and determine the involvement of NF-κB inhibition in plumbagin-mediated cell invasion suppression. In the present research we showed that plumbagin inhibited the transcriptional activity of NF-κB in HER2-positive breast cancer cells. The suppression of NF-κB activation corresponded with the inhibition of NF-κB p65 phosphorylation and downregulation of NF-κB-regulated matrix metalloproteinase 9 (MMP-9) expression. Plumbagin suppressed the invasion of HER2-overexpressing breast cancer cells and the inhibition of cell invasion was associated with the ability of plumbagin to inhibit NF-κB transcriptional activity. The silencing of NF-κB p65 increased the sensitivity of HER2-overexpressing breast cancer cells to plumbagin-induced cell invasion inhibition. NF-κB inhibition was associated with IκB kinase α (IKKα) activity suppression and inhibition of IκBα phosphorylation and degradation. The knockdown of IKKα resulted in increased sensitivity of HER2-positive cells to plumbagin-induced suppression of NF-κB transcriptional activity and expression of MMP-9. In conclusion, plumbagin inhibits the invasion of HER2-overexpressing breast cancer cells through the inhibition of IKKα-mediated NF-κB activation and downregulation of NF-κB-regulated MMP-9 expression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Down-Regulation; Female; Humans; I-kappa B Kinase; Matrix Metalloproteinase 9; Naphthoquinones; NF-kappaB-Inducing Kinase; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, ErbB-2; RNA Interference; RNA, Small Interfering; Transcription Factor RelA; Transcription, Genetic

Plumbagin is a quinonoid constituent extracted from Plumbago genus, and it exhibits diverse pharmacological effects. This study thoroughly investigated the effects of plumbagin on thioacetamide-induced acute and chronic liver injury. Results shown that plumbagin increased survival rate, reduced liver congestion and inflammation, and decreased macrophages and neutrophils in the fulminant hepatic failure model, and remarkably diminished liver fibrosis and inflammation in the chronic liver injury model. Furthermore, plumbagin significantly suppress the HSCs/myofibroblasts activation by reduced expression of markers α-SMA and COL-1/3, and reduced macrophage in liver. In the in vitro study, plumbagin induced apoptosis and suppressed the proliferation of LX-2 cells (human HSCs). Plumbagin treatment increased AMPK phosphorylation and attenuated NF-κB, STAT3, and Akt/mTOR signals in LX-2 cells, while SMAD2 phosphorylation was not changed. Noticeably, plumbagin promoted AMPK binding to p300 which is a cofactor of SMAD complex, this may further competitively decreases the p300/SMAD complex initiated transcription of COL-1/3 and α-SMA. Additionally, plumbagin hampered inflammation related NF-κB signal in RAW 264.7 cells. In conclusion, these findings indicate that plumbagin may be a powerful drug candidate to protect the liver from acute and chronic damage by inhibiting inflammation and collagen production.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Apoptosis; Chemical and Drug Induced Liver Injury; Collagen Type I; Collagen Type III; Cytoprotection; Female; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis, Experimental; Liver Failure, Acute; Liver Regeneration; Mice; Mice, Inbred ICR; Myofibroblasts; Naphthoquinones; NF-kappa B; p300-CBP Transcription Factors; Phosphorylation; Proto-Oncogene Proteins c-akt; RAW 264.7 Cells; Signal Transduction; STAT3 Transcription Factor; Thioacetamide; TOR Serine-Threonine Kinases

The antimalarial activity of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naturally occurring naphthoquinone widely distributed in the Plumbaginaceae family has previously been demonstrated in vitro (good activity) and in vivo (weak activity). The aim of the study was to investigate the pharmacokinetic profile following a single oral dosing to explain inconsistency of results of the in vitro and in vivo antimalarial activities. In addition, toxicity profiles and potential of modulation of cytochrome P450 enzymes (CYP1A2 and CYP3A11) were also investigated.. The pharmacokinetics and toxicity of plumbagin were investigated in rats. The propensity of plumbagin to modulate the mRNA expression and activities of the two inducible forms of hepatic drug metabolizing enzyme cytochrome P450 (CYP450), i.e., CYP1A2 and CYP3A11, was investigated using microsomes prepared from mouse livers.. Acute and subacute toxicity tests indicate low toxicity of plumbagin with maximum tolerated doses of 150 (single oral dose) and 25 (daily doses for 28 days) mg/kg body weight, respectively. The pharmacokinetic profile of plumbagin following a single oral dose of 100 mg/kg body weight suggests that delayed absorption and short residence time (median values of time to maximal concentration and elimination half-life = 9.63 and 5.0 h, respectively) in plasma. Plumbagin did not modulate mRNA expression and activities of CYP1A2 and CYP3A11.. Plumbagin was well tolerated following oral dose administration in rats. Pharmacokinetic property of this compound may be a limiting factor that explains the weak antimalarial activity of plumbagin observed in animal models. Potential metabolic interaction with co-administered drugs that are metabolized by CYP1A2 or CYP3A11 are unlikely.

Topics: Administration, Oral; Animals; Antimalarials; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Interactions; Female; Male; Mice; Mice, Inbred ICR; Naphthoquinones; Rats; Rats, Wistar

Plumbagin was isolated and characterized from the roots of Plumbago zeylanica using chromatography: TLC, Column chromatogram, HPLC, FTIR and

Topics: Acetylcholinesterase; Animals; Anopheles; Cytochrome P-450 Enzyme System; Esterases; Female; Gastrointestinal Tract; Glutathione Transferase; Insect Proteins; Insect Repellents; Larva; Malaria; Mosquito Vectors; Muscles; Naphthoquinones; Superoxide Dismutase

Cancer is a major public health concern globally and chemotherapy remains the principal mode of the treatment of various malignant diseases.. This study was designed to investigate the cytotoxicity of 14 naturally occurring quinones including; 3 anthraquinones, 1 naphthoquinone and 10 benzoquinones against 6 human carcinoma cell lines and normal CRL2120 fibroblasts. The neutral red uptake (NR) assay was used to evaluate the cytotoxicity of the compounds, whilst caspase-Glo assay was used to detect caspases activation. Cell cycle and mitochondrial membrane potential (MMP) were all analyzed via flow cytometry meanwhile levels of reactive oxygen species (ROS) were measured by spectrophotometry.. Anthraquinone: emodin (2), naphthoquinone: plumbagin (4), and benzoquinones: rapanone (9), 2,5-dihydroxy-3-pentadecyl-2,5-cyclohexadiene-1,4-dione (10), 5-O-methylembelin (11), 1,2,4,5-tetraacetate-3-methyl-6-(14-nonadecenyl)-cyclohexadi-2,5-diene (13), as well as doxorubicin displayed interesting activities with IC. The tested natural products and mostly 2, 4, 9, 10, 11 and 13 are potential cytotoxic compounds that deserve more investigations towards developing novel antiproliferative drugs against human carcinoma.

Topics: A549 Cells; Antineoplastic Agents, Phytogenic; Apoptosis; Benzoquinones; Caco-2 Cells; Cell Cycle; Cell Line, Tumor; Hep G2 Cells; Humans; Kenya; MCF-7 Cells; Naphthoquinones; Plant Extracts; Quinones

Spinal cord injury causes post-traumatic degeneration through series of biochemical events. This study aims to evaluate the possible protective mechanism of Plumbagin against Spinal cord injury induced oxidative stress and inflammation. Plumbagin is a potent antioxidant and shows anti-carcinogenic, anti-inflammatory and analgesic activities. However, its exact molecular mechanism of action has yet to be explored.. We tested the effects of Plumbagin on spinal cord injury induced ROS generation and lipid peroxidation content in wistar rats. Additionally, the expression of 2 important transcription factors NF-κB and Nrf-2 was investigated.. Plumbagin treatment significantly ameliorated oxidative stress through inhibition of ROS and lipid peroxidation with a concomitant increase in antioxidant status. Western blot analysis revealed enhanced nuclear levels of Nrf-2, while NF-κB expression was suppressed during Plumbagin administration. Enzyme linked immunosorbent assay for pro-inflammatory cytokines (TNF-α, IL-1β) showed a significant downregulation followed by Plumbagin treatment in spinal cord injury rats.. Taken together, the data suggests potential and novel role of Plumbagin in cytoprotection by modulating NF-κB and Nrf-2 levels against spinal cord injury.

Topics: Animals; Antioxidants; Inflammation; Male; Naphthoquinones; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Protective Agents; Rats; Spinal Cord Injuries; Up-Regulation

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a bicyclic naphthoquinone naturally distributed among Plumbago species, has been reported to have antimicrobial activity against a wide range of microorganisms. In this study, plumbagin was examined for its combinatory antimicrobial effect with tetracycline or oxacillin against nine strains of Staphylococcus aureus, including its methicillin- and multidrug-resistant strains. Minimum inhibitory concentrations were determined through the broth microdilution method, whereas the combinatory effect was evaluated according to the sum of fractional inhibitory concentration (ΣFIC) indices. Additive interactions were obtained for both combinations against most of the strains tested. Synergy was obtained for combination with oxacillin against two out of seven strains (ΣFIC range 0.273-0.281), both were methicillin resistant. Our results proved plumbagin as a compound suitable for anti-Staphylococcal combinatory testing. Moreover, to the best of our knowledge, this is the first report of plumbagin synergy with oxacillin against S. aureus strains, including its resistant forms.

Topics: Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Drug Synergism; Microbial Sensitivity Tests; Naphthoquinones; Oxacillin; Staphylococcus aureus; Tetracycline

An optimized protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Plumbago zeylanica L. was developed through selection of suitable explant type and the bacterial strain. The infection of internodal explants from an in vivo plant and leaves of in vitro origin with the A4 strain resulted in the emergence of hairy roots at a transformation frequency of 86.33 and 42.33 %, respectively. Independent transformed root somaclones (rhizoclones) capable of sustained growth were maintained under a low illumination in auxin-free agar-solidified Murashige and Skoog (MS) medium through subcultures at periodic intervals. The presence of pRi T L-DNA rolB or rolC genes and pRi T R-DNA mas2 gene in the transformed rhizoclone genome was ascertained by PCR amplification. Concentrations and type of carbon source, auxin and media strength were optimized for root biomass growth. Five independent rhizoclones each from A4- and LBA9402-transformed root lines were studied for their plumbagin accumulation at different growth phases, using HPLC analysis. The potential for plumbagin biosynthesis was expressed in all the tested rhizoclones, although distinct inter-clonal variations were noted. It was evident that maturation of hairy roots was more important for plumbagin accumulation; slow-growing and early-maturing rhizoclones accumulated more plumbagin compared to fast-growing and late-maturing rhizoclones. A4-induced rhizoclone HRA2B5 was identified as the most superior clone with a higher plumbagin yield potential in comparison with other tested hairy root clones, in vitro-grown non-transformed roots and in vivo roots of naturally occurring P. zeylanica.

Topics: Biomass; Biosynthetic Pathways; Carbohydrates; Chromatography, High Pressure Liquid; Clone Cells; Culture Media; DNA, Bacterial; Genes, Plant; Indoleacetic Acids; Kinetics; Limit of Detection; Naphthoquinones; Plant Roots; Plants, Genetically Modified; Plasmids; Plumbaginaceae; Polymerase Chain Reaction; Reproducibility of Results; Transformation, Genetic

Maintaining cellular redox homeostasis is imperative for the survival and normal functioning of cells. This study describes the role and regulation of MAPKinases in oxidative stress mediated apoptosis. Plumbagin, a vitamin K3 analog and a pro-oxidant, was employed and it induced apoptosis in both mouse and human T-cell lymphoma cell lines via increased oxidative stress, caspase activity and loss of mitochondrial membrane potential. The pro-oxidant and cytotoxic effects of plumbagin were sensitive to antioxidants indicating a decisive role of cellular redox balance. Plumbagin induced persistent activation of JNK and pharmacological inhibition as well as shRNA-mediated JNK knock-down rescued cells from plumbagin-induced apoptosis. Further, plumbagin induced cytochrome c release, FasL expression and Bax levels via activation of JNK pathway. Exposure of lymphoma cells to plumbagin led to inhibition of total and specific phosphatase activity, increased total protein S-glutathionylation and induced glutathionylation of dual specific phosphatase- 1 and 4 (MKP-1 and MKP-2). The in vivo anti-tumor efficacy of plumbagin was demonstrated using a mouse model. In conclusion, oxidative stress mediated tumor cytotoxicity operates through sustained JNK activation via a novel redox-mediated regulation of MKP-1 and MKP-2.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Disease Models, Animal; Dual Specificity Phosphatase 1; Dual-Specificity Phosphatases; Enzyme Activation; Glutathione; Humans; Jurkat Cells; Lymphoma, T-Cell; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase Phosphatases; Models, Molecular; Naphthoquinones; Oxidative Stress; Phosphorylation; Random Allocation

Plumbagin, a natural quinonoid constituent isolated from the root of medicinal plant Plumbago zeylanica L, has exhibited anti-tumor and anti-proliferative activities in various tumor cell lines as well as in animal tumor models. However, its anticancer effects and the mechanisms underlying its suppression of glioma cell growth have not been elucidated. Oncogenic transcription factor Forkhead Box M1 (FOXM1) has garnered particular interest in recent years as a potential target for the prevention and/or therapeutic intervention in glioma, nevertheless, less information is currently available regarding FOXM1 inhibitor. Here, we reported that plumbagin could effectively inhibit cell proliferation, migration and invasion and induce apoptosis of glioma cells. Cell cycle assay showed that plumbagin induced G2/M arrest. Interestingly, we found that plumbagin decreased the expression of FOXM1 both at mRNA level and protein level. Plumbagin also inhibited the transactivation ability of FOXM1, resulting in down-regulating the expression of FOXM1 downstream target genes, such as cyclin D1, Cdc25B, survivin, and increasing the expression of p21(CIP1) and p27(KIP1). Most importantly, down-regulation of FOXM1 by siFOXM1 transfection enhanced plumbagin-induced change in viability. On the contrary, over-expression of FOXM1 by cDNA transfection reduced plumbagin-induced glioma cell growth inhibition. These results suggest that plumbagin exhibits its anticancer activity partially by inactivation of FOXM1 signaling pathway in glioma cells. Our findings indicate that plumbagin may be considered as a potential natural FOXM1 inhibitor, which could contribute to the development of new anticancer agent for therapy of gliomas.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Naphthoquinones; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection

Plumbagin has received extensive attention as a promising anticancer drug. Therefore, we investigated the binding and anticancer properties of plumbagin with human serum albumin. Fluorescence results demonstrated that plumbagin interacts with human serum albumin, although its binding affinity may be affected to various extents by different compounds. The human serum albumin-plumbagin complex structure revealed that plumbagin binds to the hydrophobic cavity in the IIA subdomain of human serum albumin through hydrogen bonding and hydrophobic interactions. The plumbagin-human serum albumin complex enhances cytotoxicity by 2- to 3-fold particularly in cancer cells but has no effect on normal cells in vitro. Compared with the unbound drug, the human serum albumin-plumbagin complex promotes HeLa cell apoptosis and has a stronger capacity for cell cycle arrest at the G2/M phase of HeLa cells. In conclusion, this study contributes to the rational design and development of plumbagin-based drugs and a drug-human serum albumin delivery system.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Crystallography, X-Ray; Drug Delivery Systems; Drug Design; HeLa Cells; Humans; Kinetics; Models, Molecular; Naphthoquinones; Protein Binding; Serum Albumin; Structure-Activity Relationship

Infectious diseases and increasing antibiotic resistance among diverse classes of microbes are global health concerns and a prime focus of omics systems science applications in novel drug discovery. Plumbagin is a plant-derived naphthoquinone, a natural product that exhibits antibacterial activity against gram-positive bacteria. In the present study, we investigated the antimicrobial effects of plumbagin against Bacillus subtilis using two complementary proteomics techniques: two-dimensional electrophoresis (2-DE) and isobaric tag for relative and absolute quantification (iTRAQ). Comparative quantitative proteomics analysis of plumbagin treated and untreated control samples identified differential expression of 230 proteins (1% FDR, 1.5 fold-change and ≥2 peptides) in B. subtilis after plumbagin treatment. Pathway analysis involving the differentially expressed proteins suggested that plumbagin effectively increases heme and protein biosynthesis, whereas fatty acid synthesis was significantly reduced. Gene expression and metabolic activity assays further corroborated the proteomics findings. We anticipate that plumbagin blocks the cell division by altering the membrane permeability required for energy generation. This is the first report, to the best of our knowledge, offering new insights, at proteome level, for the putative mode(s) of action of plumbagin and attendant cellular targets in B. subtilis. The findings also suggest new ways forward for the modern omics-guided drug target discovery, building on traditional plant medicine.

Topics: Bacillus subtilis; Naphthoquinones; Proteomics; Real-Time Polymerase Chain Reaction

Plumbagin (PL), a naturally occurring quinoid, exerts antitumoral effects in diverse types of cancer cells. However, the effect of PL on tumor cell proliferation in oral squamous cell carcinoma (OSCC) remains poorly understood. In this study, we assessed the efficacy of PL, in human OSCC cells.. The effect of PL on the cell growth and apoptosis of OSCC cell lines was evaluated using MTT and Annexin V assays, respectively. The effect of PL on mitochondrial membrane potential loss and reactive oxygen species (ROS) generation was evaluated using flow cytometry analysis.. MTT assay showed that PL dose-dependently suppressed OSCC cell growth, with IC50 values ranging from 3.87 to 14.6 μM. Flow cytometry analysis revealed that PL treatment resulted in a significant decrease in mitochondrial membrane potential and an increase in the number of apoptotic cells. Notably, ROS generation was significantly elevated after PL treatment. Furthermore, a ROS scavenger, N-acetylcysteine (NAC), clearly suppressed the decrease in mitochondrial membrane potential, increase of caspase-3/7 activity, and apoptosis after PL treatment.. This study provides the considerable evidence of the tumor-suppressive effect of PL, thereby highlighting its therapeutic potential for OSCC treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Mouth Neoplasms; Naphthoquinones

Plumbagin (PLB) has exhibited a potent anticancer effect in preclinical studies, but the molecular interactome remains elusive. This study aimed to compare the quantitative proteomic responses to PLB treatment in human prostate cancer PC-3 and DU145 cells using the approach of stable-isotope labeling by amino acids in cell culture (SILAC). The data were finally validated using Western blot assay. First, the bioinformatic analysis predicted that PLB could interact with 78 proteins that were involved in cell proliferation and apoptosis, immunity, and signal transduction. Our quantitative proteomic study using SILAC revealed that there were at least 1,225 and 267 proteins interacting with PLB and there were 341 and 107 signaling pathways and cellular functions potentially regulated by PLB in PC-3 and DU145 cells, respectively. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT), and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the p

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Binding Sites; Biomarkers, Tumor; Blotting, Western; Cell Cycle; Cell Line, Tumor; Computational Biology; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Humans; Male; Molecular Docking Simulation; Molecular Structure; Naphthoquinones; Neoplasm Proteins; Prostatic Neoplasms; Protein Conformation; Protein Interaction Maps; Proteomics; Reproducibility of Results; Signal Transduction; Structure-Activity Relationship; Time Factors

Prostate cancer continues to remain the most common cancer and the second leading cause of cancer-related deaths in American males. The Pten deletions and/or mutations are frequently observed in both primary prostate cancers and metastatic prostate tissue samples. Pten deletion in prostate epithelium in mice results in prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma. The Pten conditional knockout mice [(Pten-loxp/loxp:PB-Cre4(+)) (Pten-KO)] provide a unique preclinical model to evaluate agents for efficacy for both the prevention and treatment of prostate cancer. We present here for the first time that dietary plumbagin, a medicinal plant-derived naphthoquinone (200 or 500 ppm) inhibits tumor development in intact as well as castrated Pten-KO mice. Plumbagin has shown no signs of toxicity at either of these doses. Plumbagin treatment resulted in a decrease expression of PKCε, AKT, Stat3, and COX2 compared with the control mice. Plumbagin treatment also inhibited the expression of vimentin and slug, the markers of epithelial-to-mesenchymal transition (EMT) in prostate tumors. In summary, the results indicate that dietary plumbagin inhibits growth of both primary and castration-resistant prostate cancer (CRPC) in Pten-KO mice, possibly via inhibition of PKCε, Stat3, AKT, and EMT markers (vimentin and slug), which are linked to the induction and progression of prostate cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Phytogenic; Biomarkers; Carcinogenesis; Epithelial-Mesenchymal Transition; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naphthoquinones; Orchiectomy; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Protein Kinase C-epsilon; PTEN Phosphohydrolase; Signal Transduction; STAT3 Transcription Factor

Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5'-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/mammalian target of rapamycin and p38 MAPK mediated pathways.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis Regulatory Proteins; Autophagy; Cell Cycle Checkpoints; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Epithelial-Mesenchymal Transition; Humans; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Phenotype; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases

We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase (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 displayed enhanced mammalian pol inhibition, with plumbagin conjugated to docosahexaenoic acid (C22:6-acyl plumbagin) exhibiting the strongest inhibition of pol λ among the ten 5-O-acyl plumbagins synthesized. C22:6-acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols or DNA metabolic enzymes tested. The inhibition of pol λ, a DNA repair/recombination pol, by these compounds was significantly correlated with both their suppression of lipopolysaccharide (LPS) induced tumor necrosis factor-α (TNF-α) production by mouse RAW264.7 macrophages and the reduction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the mouse ear. These data indicate that 5-O-acyl plumbagins act as anti-inflammatory agents by inhibiting mammalian pol λ. These results further suggest that C22:6-acyl plumbagin is a promising anti-inflammatory candidate and that acylation could be an effective chemical modification to improve the anti-inflammatory activity of vitamin K3 derivatives, such as plumbagin.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; DNA Polymerase beta; Escherichia coli Proteins; Fatty Acids; Gene Knockdown Techniques; Humans; Inflammation; Lipopolysaccharides; Macrophages; Mice; Naphthoquinones; Plant Proteins; Rats; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Stable nano-formulation of Plumbagin nanoparticles from Plumbago zeylanica root extract was explored as a potential natural drug against prostate cancer. Size and morphology analysis by DLS, SEM and AFM revealed the average size of nanoparticles prepared was 100±50nm. In vitro cytotoxicity showed concentration and time dependent toxicity on prostate cancer cells. However, plumbagin crude extract found to be highly toxic to normal cells when compared to plumbagin nanoformulation, thus confirming nano plumbagin cytocompatibility with normal cells and dose dependent toxicity to prostate cells. In vitro hemolysis assay confirmed the blood biocompatibility of the plumbagin nanoparticles. In wound healing assay, plumbagin nanoparticles provided clues that it might play an important role in the anti-migration of prostate cancer cells. DNA fragmentation revealed that partial apoptosis induction by plumbagin nanoparticles could be expected as a potent anti-cancer effect towards prostate cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; DNA Fragmentation; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Male; Mice; Nanoparticles; Naphthoquinones; NIH 3T3 Cells; Particle Size; Plant Extracts; Plumbaginaceae; Prostatic Neoplasms; Time Factors

A recent study reported that plumbagin downregulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various antitumor effects in multiple myeloma cells. We aimed in this in vitro study to demonstrate the inhibition of JAK2/STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in the MKN-28 gastric cancer cell line. We performed western blot analysis to measure SHP1, phosphor-JAK2/STAT3 level, and observed that plumbagin induced SHP1 expression and simultaneously downregulated phosphor-JAK2/STAT3 in MKN-28 cells, with negative SHP1 expression. This effect was consistent when JAK2/STAT3 signaling was activated by interleukin-6 (IL-6), and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, vascular endothelial growth factor (VEGF)-1, Bcl-xL, survivin and matrix metalloproteinase-9 (MMP-9), known target products of STAT3 activation in gastric carcinogenesis by reverse transcription-polymerase chain reaction (RT-PCR). Several functional studies such as water soluble tetrazolium salt-1 (WST-1) assay, wound closure assay, Matrigel invasion assay and Annexin V assay were also performed, and we validated the functional effect of plumbagin for inhibition of cell proliferation, migration and invasion, and induction of apoptosis. Collectively, our findings suggest that plumbagin is a potential regulator of cellular growth, migration, invasion and apoptosis by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; Naphthoquinones; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Signal Transduction; STAT3 Transcription Factor; Stomach Neoplasms

Our previous studies have shown that plumbagin effectively inhibits hepatic stellate cell (HSC) proliferation. Thus, plumbagin-mediated anti-fibrotic effects in vivo merit further investigation.. We used rat models to assess the potential benefits of plumbagin against CCl₄-induced liver fibrosis.. The results showed that plumbagin lowered the serum concentrations of liver functional enzymes (ALT, AST, ALB, TBIL) in CCl₄-fibrotic rats while reducing inflammatory cytokine levels (IL-6, TNF-α). As reflected in pathological examinations, rats that were administered plumbagin showed decreased collagen markers (HA, LN, PCIII and CIV) in liver tissues and improved hepatocellular impairments. In addition, plumbagin contributed to down-regulating NF-κB and TLR-4 mRNA in CCl₄-lesioned livers. As revealed in the immunohistochemical assay, plumbagin-administered rats showed reduced levels of α-SMA and TNF-α immunoreactive cells in liver tissue.. Collectively, these findings offer appealing evidence that plumbagin may serve as an anti-fibrotic medication through inactivating the NF-κB/TLR-4 pathway that is associated with inflammatory reactions, thereby mitigating liver fibrosis.

Topics: Actins; Alanine Transaminase; Animals; Aspartate Aminotransferases; Body Weight; Carbon Tetrachloride; Collagen; Down-Regulation; Fibrinolytic Agents; Interleukin-6; Liver; Liver Cirrhosis, Experimental; Male; Naphthoquinones; NF-kappa B; Rats; Rats, Sprague-Dawley; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Plumbagin (PLB) has been shown to have anticancer activities in animal models, but the role of PLB in prostate cancer treatment is unclear. This study aimed to investigate the effects of PLB on apoptosis and autophagy and the underlying mechanisms in human prostate cancer cell lines PC-3 and DU145. Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells. PLB induced mitochondria-mediated apoptosis and autophagy in concentration- and time-dependent manners in both PC-3 and DU145 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways and activation of 5'-AMP-dependent kinase (AMPK) as indicated by their altered phosphorylation, contributing to the pro-autophagic activity of PLB. Modulation of autophagy altered basal and PLB-induced apoptosis in both cell lines. Furthermore, PLB downregulated sirtuin 1 (Sirt1), and inhibition of Sirt1 enhanced autophagy, whereas the induction of Sirt1 abolished PLB-induced autophagy in PC-3 and DU145 cells. In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines. Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells. These findings indicate that PLB promotes apoptosis and autophagy in prostate cancer cells via Sirt1- and PI3K/Akt/mTOR-mediated pathways with contribution from AMPK-, p38 MAPK-, visfatin-, and ROS-associated pathways.

Topics: Apoptosis; Autophagy; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Male; Mitochondria; Molecular Structure; Naphthoquinones; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirtuin 1; Structure-Activity Relationship; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PLB), a naturally occurring naphthoquinone isolated from the roots of Plumbaginaceae plants, has been reported to possess anticancer activities in both in vitro and in vivo studies, but the effect of PLB on tongue squamous cell carcinoma (TSCC) is not fully understood. This study aimed to investigate the effects of PLB on cell cycle distribution, apoptosis, and autophagy, and the underlying mechanisms in the human TSCC cell line SCC25. The results have revealed that PLB exerted potent inducing effects on cell cycle arrest, apoptosis, and autophagy in SCC25 cells. PLB arrested SCC25 cells at the G2/M phase in a concentration- and time-dependent manner with a decrease in the expression level of cell division cycle protein 2 homolog (Cdc2) and cyclin B1 and increase in the expression level of p21 Waf1/Cip1, p27 Kip1, and p53 in SCC25 cells. PLB markedly induced apoptosis and autophagy in SCC25 cells. PLB decreased the expression of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) while increasing the expression level of the pro-apoptotic protein Bcl-2-associated X protein (Bax) in SCC25 cells. Furthermore, PLB inhibited phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β), and p38 mitogen-activated protein kinase (p38 MAPK) pathways as indicated by the alteration in the ratio of phosphorylation level over total protein expression level, contributing to the autophagy inducing effect. In addition, we found that wortmannin (a PI3K inhibitor) and SB202190 (a selective inhibitor of p38 MAPK) strikingly enhanced PLB-induced autophagy in SCC25 cells, suggesting the involvement of PI3K- and p38 MAPK-mediated signaling pathways. Moreover, PLB induced intracellular reactive oxygen species (ROS) generation and this effect was attenuated by l-glutathione (GSH) and n-acetyl-l-cysteine (NAC). Taken together, these results indicate that PLB promotes cellular apoptosis and autophagy in TSCC cells involving p38 MAPK- and PI3K/Akt/mTOR-mediated pathways with contribution from the GSK3β and ROS-mediated pathways.

Topics: Apoptosis; Autophagy; Carcinoma, Squamous Cell; Cell Division; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Humans; M Phase Cell Cycle Checkpoints; MAP Kinase Signaling System; Molecular Structure; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Structure-Activity Relationship; Tongue Neoplasms; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFβ in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFβ. We previously showed that Src, JNK, and Smad3 mediate TGFβ1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGFβ. In this study, we demonstrated that TGFβ1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFβ1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFβ1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFβ1-induced NOX4 expression in HGFs.

Topics: Acetylcysteine; Cell Culture Techniques; Cells, Cultured; Connective Tissue Growth Factor; Curcumin; Enzyme Inhibitors; Fibroblasts; Free Radical Scavengers; Gene Silencing; Gingiva; Gingival Overgrowth; Humans; MAP Kinase Signaling System; NADPH Oxidase 4; NADPH Oxidases; Naphthoquinones; Oxidation-Reduction; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Smad3 Protein; src-Family Kinases; Superoxides; Transforming Growth Factor beta1

Long-term and high-dose glucocorticoids (GCs) supplementation has been linked to osteoporosis. In this study, we studied the protective role of plumbagin against GC-induced cell damage in MC3T3-E1 cells. The effect of dexamethasone (DEX) and plumbagin on cell viability was determined. DEX showed as IC-50 value of 95 μM. Further, 10 μM plumbagin treatment effectively ameliorated DEX-induced cell death by increasing the cell viability to 92 %. A further effect of plumbagin on DEX-induced oxidative stress was determined through reactive oxygen species (ROS) level, lipid peroxide content, and antioxidant status. Nrf-2 nuclear localization was analyzed through immunofluorescence. Protein expression of redox regulator Nrf-2 and their target genes HO-1 and NQO1 and osteogenic markers (OCN, OPN Runx-2) were determined by Western blot. Apoptotic effect was analyzed by mitochondrial membrane potential and caspase activities (3, 8, and 9). The results showed that DEX treatment showed a significant increase in oxidative stress through increased ROS levels and downregulation of cytoprotective antioxidant proteins and antioxidant enzyme activities. Further DEX treatment downregulated the osteogenic markers and upregulated apoptosis through decreased mitochondrial membrane potential and upregulation of caspase activities. Plumbagin treatment significantly reversed the levels of oxidative stress and apoptotic markers and protected against DEX-induced cell damage. Further, plumbagin treatment significantly improved the expression of osteogenic markers compared to DEX treatment. In conclusion, the present study shows that plumbagin offers significant protective role against DEX-induced cellular damage via regulating oxidative stress, apoptosis, and osteogenic markers.

Topics: Animals; Apoptosis; Caspases; Cell Line; Cell Survival; Core Binding Factor Alpha 1 Subunit; Dexamethasone; Enzyme-Linked Immunosorbent Assay; Heme Oxygenase-1; Lipid Peroxidation; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; NF-E2-Related Factor 2; Osteocalcin; Osteopontin; Osteoporosis; Protective Agents; Superoxide Dismutase

Chemoresistance in cancer has previously been attributed to gene mutations or deficiency. Caspase mutations or Bax deficiency can lead to resistance to cancer drugs. We recently demonstrated that Bak initiates a caspase/Bax-independent cell death pathway. We show that Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone that is known to have anti-tumor activity in a variety of models, induces caspase-independent cell death in HCT116 Bax knockout (KO) or MCF-7 Bax knockdown (KD) cells that express wild-type (WT) Bak. The re-expression of Bax in HCT116 Bax KO cells fails to enhance the PL-induced cell death. Additionally, Bak knockdown by shRNA efficiently attenuates PL-induced cell death. These results suggest that PL-induced cell death depends primarily on Bak, not Bax, in these cells. Further experimentation demonstrated that p53 Ser15 phosphorylation and mitochondrial translocation mediated Bak activation and subsequent cell death. Knockdown of p53 or a p53 Ser15 mutant significantly inhibited p53 mitochondrial translocation and cell death. Furthermore, we found that Akt mediated p53 phosphorylation and the subsequent mitochondrial accumulation. Taken together, our data elaborate the role of Bak in caspase/Bax-independent cell death and suggest that PL may be an effective agent for overcoming chemoresistance in cancer cells with dysfunctional caspases.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Drug Resistance, Neoplasm; Fluorescent Antibody Technique; Gene Knockdown Techniques; Gene Knockout Techniques; Humans; Mitochondria; Naphthoquinones; Phosphorylation; RNA, Small Interfering; Transfection; Tumor Suppressor Protein p53

Antioxidants have not reduced the burden of cardiovascular disease, and current evidence suggests a beneficial role of oxidative stress, via NADPH oxidase (Nox) upregulation, in endothelial function. Homocysteine thiolactone (HcyT) induces blood vessel dysfunction and this correlates with increased vascular oxidative stress. This study aimed to determine if pharmacological inhibition of Nox could impair HcyT induced blood vessel dysfunction. Abdominal aorta were excised from New Zealand White rabbits (n = 6), cut into rings and sequentially mounted in organ baths. Rings were preincubated with 0.55 μmol/L homocysteine thiolactone for 1 h, or combinations of putative Nox inhibitors (plumbagin for Nox4, gp91ds-tat for Nox2, and ML090 for Nox1), 30 min prior to the addition of HcyT, followed by a dose response curve to acetylcholine on phenylephrine preconstricted rings. Plumbagin, ML090 + gp91ds-tat and HcyT reduced responses to acetylcholine, and Plumbagin + Hcyt caused constriction to acetylcholine, which was normalised to plumbagin by ML090. Plumbagin + ML090 or plumbagin + gp91ds-tat completely impaired the effect of acetylcholine. ML090 inhibited the effect of HcyT on reduced response to acetylcholine, whereas gp91ds-tat had no effect. This study concludes that inhibition of Nox1 prevents, whereas inhibition of Nox4 worsens, acetylcholine induced blood vessel relaxation caused by HcyT, while Nox2 inhibition has no effect. However combinations of Nox inhibitors worsen acetylcholine induced blood vessel relaxation. These results suggest that there is cross-talk between Nox isoforms during physiological and pathophysiological processes.

Topics: Animals; Aorta, Abdominal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glycoproteins; Homocysteine; Male; NADPH Oxidases; Naphthoquinones; Quinoxalines; Rabbits

Plumbagin is a natural compound that is isolated from the root of the medicinal plant Plumbago zeylanica L. Based on a previous in vitro study performed by our group, which demonstrated the effectiveness of plumbagin against glioma cells, we further ascertained whether plumbagin exhibits the same effectiveness against glioma cell xenografts in nude mice. Our results revealed that tumor volume was reduced by 54.48% in the plumbagin-treated group compared with the controls. Furthermore, there were no obvious signs of toxicity as assessed by the organ sizes and cell morphologies of the mice that were treated with plumbagin. Immunofluorescence assays further revealed that plumbagin significantly inhibited glioma cell proliferation and induced cell apoptosis. Importantly, we also determined that the expressions of FOXM1 and its downstream target effectors, including cyclin D1 and Cdc25B, were down-regulated in the treated group, while the expressions of p21 and p27 were increased; the latter findings corroborate the results of our previous in vitro study. Taken together, these findings indicate that plumbagin may be a natural downregulator of FOXM1 with potential therapeutic effectiveness for the treatment of gliomas.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression; Glioma; Humans; Mice, Nude; Molecular Targeted Therapy; Naphthoquinones; Neoplasm Transplantation; Phytotherapy; Plumbaginaceae

The expression levels of anoctamin 1 (ANO1, TMEM16A), a calcium-activated chloride channel (CaCC), are significantly increased in several tumors, and inhibition of ANO1 is known to reduce cell proliferation and migration. Here, we performed cell-based screening of a collection of natural products and drug-like compounds to identify inhibitors of ANO1. As a result of the screening, idebenone, miconazole and plumbagin were identified as novel ANO1 inhibitors. Electrophysiological studies showed that idebenone, a synthetic analog of coenzyme Q10, completely blocked ANO1 activity in FRT cells expressing ANO1 without any effect on intracellular calcium signaling and CFTR, a cAMP-regulated chloride channel. The CaCC activities in PC-3 and CFPAC-1 cells expressing abundant endogenous ANO1 were strongly blocked by idebenone. Idebenone inhibited cell proliferation and induced apoptosis in PC-3 and CFPAC-1 cells, but not in A549 cells, which do not express ANO1. These data suggest that idebenone, a novel ANO1 inhibitor, has potential for use in cancer therapy.

Topics: Animals; Anoctamin-1; Antineoplastic Agents; Antioxidants; Apoptosis; Biological Products; Calcium Signaling; Cell Line, Tumor; Cell Proliferation; Chloride Channels; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Miconazole; Naphthoquinones; Neoplasm Proteins; Rats; Rats, Inbred F344; Ubiquinone

Zoledronic acid (ZA), a bisphosphonate, is currently used in combination with chemotherapeutic agents to suppress breast cancer cell proliferation or breast cancer-induced osteolysis. The aim of this study was to investigate the effects of ZA combined with a natural anticancer compound plumbagin (PL) against human breast cancer cells in vitro.. Human breast cancer MDA-MB-231SArfp cells were treated with ZA, PL or a combination of ZA and PL. The cell growth, apoptosis and migration were evaluated using CCK-8 assay, flow cytometry and transwell assay, respectively. The expression of apoptosis-related proteins was measured using real-time PCR and Western blotting. Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined.. PL or ZA alone caused mild cytotoxicity (the IC50 value at 24 h was 12.18 and above 100 μmol/L, respectively). However, the combination of ZA and PL caused a synergistic cytotoxicity (CI=0.26). The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively. Furthermore, PL and ZA synergistically induced apoptosis and inhibited migration of the breast cancer cells. Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1. When the breast cancer cells were transfected with specific siRNA against Notch-1, the combination of ZA and PL markedly increased the expression of Bcl-2.. Combination of ZA and PL synergistically suppresses human breast cancer MDA-MB-231SArfp cells in vitro. PL can inhibit ZA-induced activation of the Notch-1 signaling pathway and subsequently reduce the expression of Bcl-2, thus potentiating cancer cell apoptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bone Density Conservation Agents; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Diphosphonates; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Zoledronic Acid

Plumbagin is a secondary metabolite that was first identified in the Plumbago genus of plants. It is a naphthoquinone compound with anti-atherosclerosis, anticancer, anti-inflammatory, antimicrobial, contraceptive, cardiotonic, immunosuppressive, and neuroprotective activities. However, the mechanisms of plumbagin's activities are largely unknown. In this study, we examined the effect of plumbagin on HepG2 hepatocellular carcinoma cells as well as LLC lung cancer cells, SiHa cervical carcinoma cells. Plumbagin significantly decreased HepG2 cell viability in a dose-dependent manner. Additionally, treatment with plumbagin significantly increased the Bax/Bcl-2 ratio and caspase-3/7 activity. Using the similarity ensemble approach (SEA)-a state-of-the-art cheminformatic technique-we identified two previously unknown cellular targets of plumbagin: thioredoxin reductase (TrxR) and glutathione reductase (GR). This was then confirmed using protein- and cell-based assays. We found that plumbagin was directly reduced by TrxR, and that this reduction was inhibited by the TrxR inhibitor, sodium aurothiomalate (ATM). Plumbagin also decreased the activity of GR. Plumbagin, and the GR inhibitor sodium arsenite all increased intracellular reactive oxygen species (ROS) levels and this increase was significantly attenuated by pretreatment with the ROS scavenger N-acetyl-cysteine (NAC) in HepG2 cells. Plumbagin increased TrxR-1 and heme oxygenase (HO)-1 expression and pretreatment with NAC significantly attenuated the plumbagin-induced increase of TrxR-1 and HO-1 expression in HepG2 cells, LLC cells and SiHa cells. Pretreatment with NAC significantly prevented the plumbagin-induced decrease in cell viability in these cell types. In conclusion, plumbagin exerted its anticancer effect by directly interacting with TrxR and GR, and thus increasing intracellular ROS levels.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Dose-Response Relationship, Drug; Glutathione Reductase; Hep G2 Cells; Humans; Naphthoquinones; Reactive Oxygen Species; Thioredoxin-Disulfide Reductase

Plumbagin has been reported to be neuroprotective, so it might possess antidepressant activity. Therefore, the present study was designed to explore the antidepressant potential of plumbagin in unstressed and stressed mice.. Depression-like behavior was induced in Swiss male albino mice by subjecting them to unpredictable mild stress daily for 21 successive days. Plumbagin (4, 8 and 16mg/kg, po) and imipramine (15mg/kg, po) were administered for 3 successive weeks to separate groups of unstressed and stressed mice. Tail suspension test and sucrose preference test were used to evaluate antidepressant effect of the drugs.. Highest dose (16mg/kg) of plumbagin and imipramine significantly decreased immobility period of unstressed and stressed mice in tail suspension test as compared to their respective controls. These drugs significantly restored the reduced sucrose preference (%) in stressed mice. The drugs did not significantly affect locomotor activity of mice. Antidepressant-like activity of plumbagin was found to be comparable to imipramine. Plumbagin and imipramine significantly inhibited brain MAO-A activity, decreased plasma nitrite, brain malondialdehyde and catalase levels; and increased reduced glutathione levels of unstressed and stressed mice. The drugs significantly reversed stress-induced increase in plasma corticosterone levels.. Antidepressant-like activity of plumbagin in unstressed and stressed mice might be through inhibition of brain MAO-A activity and improvement of antioxidant status. Reversal of stress-induced increase in plasma corticosterone levels might also be responsible for antidepressant-like activity of plumbagin in stressed mice.

Topics: Animals; Antidepressive Agents; Antidepressive Agents, Tricyclic; Antioxidants; Behavior, Animal; Brain; Corticosterone; Depression; Hindlimb Suspension; Imipramine; Male; Mice; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Motor Activity; Naphthoquinones; Stress, Psychological

The folate (FA)-functionalized human serum albumin (HSA) carrier (FA-HSA) is promising for improving the target and efficiency of anticancer drugs. To develop FA-HSA carrier for metal anticancer drugs, we investigated anticancer properties and mechanism of FA-HSA carrier for Cu(II) complexes derived from plumbagin. The fluorescence spectroscopy and molecular docking revealed that Cu(II) complexes bind to IIA subdomain of HSA. Compared with Cu(II) complex alone, FA-HSA-metallodrug complex enhances cytotoxicity to FA-positive cancer cells (HeLa) but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent; FA-HSA-metallodrug complex has a stronger capacity for cell cycle arrest in the G2/M phase of HeLa cells, and down-regulating the expression of cyclin-dependent kinase 1 (CDK1) and cyclin B1. Moreover, FA-HSA-metallodrug complex promotes HeLa cells apoptosis through intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; CDC2 Protein Kinase; Coordination Complexes; Copper; Cyclin B1; Cyclin-Dependent Kinases; Cytochromes c; Drug Carriers; Folic Acid; HeLa Cells; Humans; MCF-7 Cells; Naphthoquinones; Serum Albumin

Colloidal silver nanoparticles (AgNPs) have attracted much attention in recent years as diagnostics and new drug delivery system in cancer medicine. To study the effects of plumbagin (PLB), a relatively non-toxic napthaquinone isolated from the roots of Plumbago indica in human cervical cancer cell line and developed a formulation to enhance its cytotoxic activities. Silver nanoparticles were synthesised by chemical reduction method and complexed with PLB. Both the AgNPs and the complex PLB-AgNPs were characterised by dynamic light scattering, high-resolution scanning electron microscopy and transmission electron microscopy. The amount of PLB and PLB-AgNPs internalised was determined by ultra-violet-visible spectrophotometer. Cell inhibition was determined by sulphorhodamine B assay. Mitotic index was determined by Wright-Giemsa staining. Apoptosis induction was assessed by western blot using cleaved poly adenosine diphosphate-ribose polymerase antibody. The scanning electron microscope analysis indicated an average particle size of 32±8 nm in diameter. Enhanced internalisation of PLB into the HeLa cells was observed in PLB-AgNPs. PLB inhibited proliferation of cells with IC50 value of about 18±0.6 µM and blocked the cells at mitosis in a concentration-dependent manner. PLB also inhibited the post-drug exposure clonogenic survival of cells and induced apoptosis. The antiproliferative, antimitotic and apoptotic activities were also found to be increased when cells were treated with PLB-AgNPs. The authors results support the idea that AgNP could be a promising and effective drug delivery system for enhanced activity of PLB in cancer treatment.

Topics: Antineoplastic Agents; Cell Proliferation; HeLa Cells; Humans; Metal Nanoparticles; Naphthoquinones; Silver

Tongue squamous cell carcinoma (TSCC) is the most common malignancy in oral and maxillofacial tumors with highly metastatic characteristics. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone; PLB), a natural naphthoquinone derived from the roots of Plumbaginaceae plants, exhibits various bioactivities, including anticancer effects. However, the potential molecular targets and underlying mechanisms of PLB in the treatment of TSCC remain elusive. This study employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic approach to investigate the molecular interactome of PLB in human TSCC cell line SCC25 and elucidate the molecular mechanisms. The proteomic data indicated that PLB inhibited cell proliferation, activated death receptor-mediated apoptotic pathway, remodeled epithelial adherens junctions pathway, and manipulated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response signaling pathway in SCC25 cells with the involvement of a number of key functional proteins. Furthermore, we verified these protein targets using Western blotting assay. The verification results showed that PLB markedly induced cell cycle arrest at G2/M phase and extrinsic apoptosis, and inhibited epithelial to mesenchymal transition (EMT) and stemness in SCC25 cells. Of note, N-acetyl-l-cysteine (NAC) and l-glutathione (GSH) abolished the effects of PLB on cell cycle arrest, apoptosis induction, EMT inhibition, and stemness attenuation in SCC25 cells. Importantly, PLB suppressed the translocation of Nrf2 from cytosol to nucleus, resulting in an inhibition in the expression of downstream targets. Taken together, these results suggest that PLB may act as a promising anticancer compound via inhibiting Nrf2-mediated oxidative stress signaling pathway in SCC25 cells. This study provides a clue to fully identify the molecular targets and decipher the underlying mechanisms of PLB in the treatment of TSCC.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; G2 Phase Cell Cycle Checkpoints; Head and Neck Neoplasms; Humans; Naphthoquinones; Neoplastic Stem Cells; NF-E2-Related Factor 2; Oxidative Stress; Protein Interaction Maps; Protein Transport; Proteomics; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Time Factors; Tongue Neoplasms

Natural plant products may possess much potential in palliative therapy and supportive strategies of current cancer treatments with lesser cytotoxicity to normal cells compared to conventional chemotherapy. In the current study, anti-cancer properties of plumbagin, a plant-derived naphthoquinone, on brain cancer cells were determined. Plumbagin treatment resulted in the induction of DNA damage, cell cycle arrest and apoptosis, followed by suppression of the colony forming ability of the brain tumour cells. These effects were substantiated by upregulation of PTEN, TNFRSF1A and downregulation of E2F1 genes, along with a drop in MDM2, cyclin B1, survivin and BCL2 protein expression. Plumbagin induced elevated levels of caspase-3/7 activity as well. For the first time, we show here that plumbagin inhibits telomerase in brain tumour cells and results in telomere shortening following chronic long-term treatment. This observation implies considerable cytotoxicity of plumbagin towards cancer cells with higher telomerase activity. Collectively, our findings suggest plumbagin as a potential chemotherapeutic phytochemical in brain tumour treatment modalities.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA Damage; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Naphthoquinones; Telomerase; Telomere; Telomere Shortening

Resistant cancer phenotype is a key obstacle in the successful therapy of prostate cancer. The primary aim of our study was to explore resistance mechanisms in the advanced type of prostate cancer cells (PC-3) and to clarify the role of autophagy in these processes. We performed time-lapse experiment (48 hours) with ROS generating plumbagin by using multimodal holographic microscope. Furthermore, we also performed the flow-cytometric analysis and the qRT-PCR gene expression analysis at 12 selected time points. TEM and confocal microscopy were used to verify the results. We found out that autophagy (namely mitophagy) is an important resistance mechanism. The major ROS producing mitochondria were coated by an autophagic membrane derived from endoplasmic reticulum and degraded. According to our results, increasing ROS resistance may be also accompanied by increased average cell size and polyploidization, which seems to be key resistance mechanism when connected with an escape from senescence. Many different types of cell-cell interactions were recorded including entosis, vesicular transfer, eating of dead or dying cells, and engulfment and cannibalism of living cells. Entosis was disclosed as a possible mechanism of polyploidization and enabled the long-term survival of cancer cells. Significantly reduced cell motility was found after the plumbagin treatment. We also found an extensive induction of pluripotency genes expression (NANOG, SOX2, and POU5F1) at the time-point of 20 hours. We suppose, that overexpression of pluripotency genes in the portion of prostate tumour cell population exposed to ROS leads to higher developmental plasticity and capability to faster respond to changes in the extracellular environment that could ultimately lead to an alteration of cell fate.

Topics: Autophagy; Cell Communication; Cell Line, Tumor; Cell Self Renewal; Cell Size; Cell Survival; Endoplasmic Reticulum; Entosis; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Male; Mitophagy; Naphthoquinones; Neoplasm Metastasis; Oxidative Stress; Principal Component Analysis; Prostatic Neoplasms; Reactive Oxygen Species; Time-Lapse Imaging

Drosera peltata Smith var. lunata (Buch.-Ham.) C. B. Clarke (DPVL) fractions and plumbagin were tested via broth microdilution techniques on Rhizopus oryzae, Aspergillus flavus, Aspergillus niger, Aspergillus oryzae, Penicillium citrinum. All of the test substances [petroleum ether, chloroform, ethyl acetate, n-butanol fraction and aqueous residue (AR)] except for the AR were active against all the tested strains. The petroleum ether fraction (PEF) was the most active (MIC = 5.86-46.88 μg/ml, MFC = 23.44-93.75 μg/ml) of the five tested substances and therefore, was selected for further analysis. Based on antifungal activity, bioactivity-guided fractionation of the PEF led to the isolation of plumbagin. The structure of plumbagin was elucidated by ¹H and ¹³C NMR. Using HPLC, DPVL was found to be a new source of plumbagin. Reversed-phase HPLC was performed using a mobile phase of water and methanol, and peaks were detected at 254 nm. Plumbagin showed a good linear relationship at concentrations ranging from 0.625 to 10 μg/ml. Both the intraday and the interday precision showed that the method was precise, with RSDs of at least 3% at different concentrations. Recovery rates ranging from 97.86 to 99.94% were observed, which indicate that the method is accurate. The specificity of the method was established by checking the peak purity of plumbagin. For six independent measurements, the average plumbagin content in DPVL was 11.05 ± 0.31 mg/g of dried material. The validated HPLC method provides a new basis for assessing DPVL quality.

Topics: Antifungal Agents; Chromatography, High Pressure Liquid; Drosera; Fungi; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Naphthoquinones; Phytochemicals

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100μg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1μg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10μg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10μg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100μg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100μg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100μg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

Topics: Animals; Antiplatyhelmintic Agents; Benzimidazoles; Buffaloes; Cattle; Cattle Diseases; Fasciola; Fascioliasis; Female; Inhibitory Concentration 50; Lymnaea; Male; Mice; Mice, Inbred ICR; Microscopy, Electron, Scanning; Naphthoquinones; Random Allocation; Triclabendazole

Plumbagin [5-hydroxy- 2-methyl-1, 4-naphthaquinone] is a well-known plant derived anticancer lead compound. Several efforts have been made to synthesize its analogs and derivatives in order to increase its anticancer potential. In the present study, plumbagin and its five derivatives have been evaluated for their antiproliferative potential in one normal and four human cancer cell lines. Treatment with derivatives resulted in dose- and time-dependent inhibition of growth of various cancer cell lines. Prescreening of compounds led us to focus our further investigations on acetyl plumbagin, which showed remarkably low toxicity towards normal BJ cells and HepG2 cells. The mechanisms of apoptosis induction were determined by APOPercentage staining, caspase-3/7 activation, reactive oxygen species production and cell cycle analysis. The modulation of apoptotic genes (p53, Mdm2, NF-kB, Bad, Bax, Bcl-2 and Casp-7) was also measured using real time PCR. The positive staining using APOPercentage dye, increased caspase-3/7 activity, increased ROS production and enhanced mRNA expression of proapoptotic genes suggested that acetyl plumbagin exhibits anticancer effects on MCF-7 cells through its apoptosis-inducing property. A key highlighting point of the study is low toxicity of acetyl plumbagin towards normal BJ cells and negligible hepatotoxicity (data based on HepG2 cell line). Overall results showed that acetyl plumbagin with reduced toxicity might have the potential to be a new lead molecule for testing against estrogen positive breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 7; Cell Cycle; Cell Survival; Female; Fibroblasts; Hep G2 Cells; Humans; Male; MCF-7 Cells; Naphthoquinones; Reactive Oxygen Species; Receptors, Estrogen; Structure-Activity Relationship

Plumbagin has been reported to modulate cellular redox status and suppress NF-κB. In the present study, we investigated the effect of plumbagin on lipopolysaccharide (LPS)-induced endotoxic shock, oxidative stress and inflammatory parameters in vitro and in vivo. Plumbagin inhibited LPS-induced nitric oxide, TNF-α, IL-6 and prostaglandin-E2 production in a concentration-dependent manner in RAW 264.7 cells without inducing any cell death. Plumbagin modulated cellular redox status in RAW cells. Plumbagin treatment significantly reduced MAPkinase and NF-κB activation in macrophages. Plumbagin prevented mice from endotoxic shock-associated mortality and decreased serum levels of pro-inflammatory markers. Plumbagin administration ameliorated LPS-induced oxidative stress in peritoneal macrophages and splenocytes. Plumbagin also attenuated endotoxic shock-associated changes in liver and lung histopathology and decreased the activation of ERK and NF-κB in liver. These findings demonstrate the efficacy of plumbagin in preventing LPS-induced endotoxemia and also provide mechanistic insights into the anti-inflammatory effects of plumbagin.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cell Line; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Inflammation Mediators; Interleukin-6; Lipopolysaccharides; Liver; Lung; Macrophages; Male; Mice; Naphthoquinones; NF-kappa B; Nitric Oxide; Oxidative Stress; Shock, Septic; Signal Transduction; Spleen; Time Factors; Tumor Necrosis Factor-alpha

Plumbagin (PLB) has shown anti-cancer activity but the mechanism is unclear. This study has found that PLB has a potent pro-apoptotic and pro-autophagic effect on A549 and H23 cells. PLB arrests cells in G2/M phase, and increases the intracellular level of reactive oxygen species in both cell lines. PLB dose-dependently induces autophagy through inhibition of PI3K/Akt/mTOR pathway as indicated by reduced phosphorylation of Akt and mTOR. Inhibition or induction of autophagy enhances PLB-induced apoptosis. There is crosstalk between PLB-induced apoptosis and autophagy. These findings indicate that PLB initiates both apoptosis and autophagy in NSCLC cells through coordinated pathways.

Topics: Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Humans; Lung Neoplasms; MAP Kinase Signaling System; Naphthoquinones; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

Plumbagin (PL) has been reported to exhibit anti-carcinogenic, anti-inflammatory and analgesic activities, but little is known about its mechanism. In this study, we investigated the anti-inflammatory property of PL and its mechanism of action. Although no significant cytotoxicity of PL was observed over the concentration range tested, PL (2.5-7.5 μM) significantly and dose-dependently suppressed the secretion of pro-inflammatory mediators and inhibited the expression of TNF-α, IL-1β, IL-6 and iNOS in LPS-stimulated RAW 264.7 cells. Furthermore, PL consistently suppressed the activity of iNOS in LPS-induced RAW 264.7 cells. To elucidate the mechanism underlying the anti-inflammatory activity of PL, we assessed the effects of PL on the MAPK pathway and the activity and expression of NF-κB. These experiments demonstrated that PL significantly reduced the luciferase activity of an NF-κB promoter reporter and p65 nuclear translocation. The LPS-induced phosphorylation of MAP kinases was also attenuated by PL; significant changes were observed in the levels of phosphorylated ERK1/2, JNK and p38 MAPK. Additionally, MAPK inhibitors confirmed the inhibitory effect of PL on the MAPK pathway. Taken together, these data suggest that PL exerts its anti-inflammatory effects by down-regulating the expression of pro-inflammatory mediators through inhibition of NF-κB and MAPK signaling in LPS-stimulated RAW 264.7 cells.

Topics: Animals; Base Sequence; Cell Line; DNA Primers; Inflammation; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; MAP Kinase Signaling System; Mice; Naphthoquinones; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphorylation; Tumor Necrosis Factor-alpha

To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice.. Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin (2, 4, or 6 mg/kg, ip) 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses.. Plumbagin (2.5-20 μmol/L) concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro (the IC50 value of inhibition of cell viability was 14.7 μmol/L). Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2-3 weeks and reduced the tumor volume by 44%-74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts.. Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Osteoclasts; Tumor Microenvironment

Plumbagin is the major active constituent in several plants including Plumbago indica Linn. (root). This compound has been shown to exhibit a wide spectrum of biological and pharmacological activities. The present study aimed to evaluate the in vitro and in vivo antimalarial activity of plumbagin including its acute and subacute toxicity in mice.. In vitro antimalarial activity of plumbagin against K1 and 3D7 Plasmodium falciparum clones were assessed using SYBR Green I based assay. In vivo antimalarial activity was investigated in Plasmodium berghei-infected mouse model (a 4-day suppressive test).. Plumbagin exhibited promising antimalarial activity with in vitro IC50 (concentration that inhibits parasite growth to 50%) against 3D7 chloroquine-sensitive P. falciparum and K1 chloroquine-resistant P. falciparum clones of 580 (270-640) and 370 (270-490) nM, respectively. Toxicity testing indicated relatively low toxicity at the dose levels up to 100 (single oral dose) and 25 (daily doses for 14 days) mg/kg body weight for acute and subacute toxicity, respectively. Chloroquine exhibited the most potent antimalarial activity in mice infected with P. berghei ANKA strain with respect to its activity on the reduction of parasitaemia on day 4 and the prolongation of survival time.. Plumbagin at the dose of 25 mg/kg body weight given for 4 days was safe and produced weak antimalarial activity. Chemical derivatization of the parent compound or preparation of modified formulation is required to improve its systemic bioavailability.

Topics: Animals; Antimalarials; Biological Availability; Body Weight; Chloroquine; Disease Models, Animal; Female; In Vitro Techniques; Malaria; Male; Mice; Mice, Inbred ICR; Naphthoquinones; Plasmodium berghei; Plasmodium falciparum; Toxicity Tests

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.

Topics: Binding Sites; Cell Line, Tumor; Cell Survival; E1A-Associated p300 Protein; Enzyme Inhibitors; HEK293 Cells; HeLa Cells; Humans; Kinetics; Lysine; Naphthoquinones; Oxidation-Reduction; Protein Structure, Tertiary; Reactive Oxygen Species; Structure-Activity Relationship; Sulfhydryl Compounds

The extraction studies and a one-step purification of the crude extract of Plumbago indica using silica-gel vacuum chromatography provided a plumbagin derivative-rich P. indica root extract (PPE). The PPE was standardised to contain total plumbagin derivatives not less than 13% w/w. Antibacterial activities of the standardised PPE and three naphthoquinones, plumbagin, elliptinone and 3,3'-biplumbagin, against Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis were evaluated by using the microdilution assay. The bactericidal activities of the PPE against these bacteria were much stronger than those of elliptinone and 3,3'-biplumbagin and almost equal to those of plumbagin. Stability of the PPE was determined under various conditions through a period of four months. The PPE was stable over a period of four months when stored as a dried powder but only in a well-closed container protected from light under 4 ± 2°C.

Topics: Anti-Bacterial Agents; Microbial Sensitivity Tests; Molecular Structure; Naphthoquinones; Plant Roots; Plumbaginaceae; Propionibacterium acnes; Staphylococcus

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

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a naphthoquinone derivative from the roots of plant Plumbago zeylanica and belongs to one of the largest and diverse groups of plant metabolites. The anticancer and antiproliferative activities of plumbagin have been observed in animal models as well as in cell cultures. Plumbagin exerts inhibitory effects on multiple cancer-signaling proteins, however, the binding mode and the molecular interactions have not yet been elucidated for most of these protein targets. The present study is the first attempt to provide structural insights into the binding mode of plumbagin to five cancer signaling proteins viz. PI3Kγ, AKT1/PKBα, Bcl-2, NF-κB, and Stat3 using molecular docking and (un)binding simulation analysis. We validated plumbagin docking to these targets with previously known important residues. The study also identified and characterized various novel interacting residues of these targets which mediate the binding of plumbagin. Moreover, the exact modes of inhibition when multiple mode of inhibition existed was also shown. Results indicated that the engaging of these important interacting residues in plumbagin binding leads to inhibition of these cancer-signaling proteins which are key players in the pathogenesis of cancer and thereby ceases the progression of the disease.

Topics: Animals; Antineoplastic Agents, Phytogenic; Class Ib Phosphatidylinositol 3-Kinase; Databases, Pharmaceutical; Humans; Ligands; Mice; Models, Molecular; Molecular Conformation; Naphthoquinones; NF-kappa B; Protein Binding; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor

Plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, has been shown to exert anti-cancer and anti-proliferative activities in vitro as well as in animal tumor models. However, the mechanism underlying its anti-tumor action still remains unclear. CRM1 is a nuclear export receptor involved in the active transport of tumor suppressors whose function is altered in cancer due to increased expression and overactive transport. We showed that CRM1 is a direct cellular target of plumbagin. The nuclei of cells incubated with plumbagin accumulated tumor-suppressor proteins and inhibited the interactions between CRM1 and these proteins. Particularly, we demonstrated that plumbagin could specifically react with the conserved Cys(528) of CRM1 but not with a Cys(528) mutant peptide through Mass spectrometric analysis. More importantly, cancer cells that are transfected with mutant CRM1 (C528S) are resistant to the inhibitory effects of plumbagin, demonstrating that the inhibition is through direct interaction with Cys(528) of CRM1. The inhibition of nuclear traffic by plumbagin may account for its therapeutic properties in cancer and inflammatory diseases. Our findings could contribute to the development of a new class of CRM1 inhibitors.

Topics: Cell Line, Tumor; Cell Nucleus; Drug Design; Exportin 1 Protein; Humans; Karyopherins; Molecular Targeted Therapy; Naphthoquinones; Neoplasms; Nuclear Proteins; Phytotherapy; Plumbaginaceae; Protein Transport; Receptors, Cytoplasmic and Nuclear; Tumor Suppressor Proteins

Plumbagin (1), a naphthoquinone, induces cell death and affects various signaling pathways in cancer cells. Wnt signaling is active constitutively in colorectal cancer and plays an important role in its progression and pathogenesis. It was hypothesized that 1 is likely to modulate Wnt signaling, and this compound was studied for its effect on this pathway in human colorectal cancer cells. Plumbagin (1) was found to downregulate Wnt signaling when assessed by a TOPFlash/FOPFlash reporter activity assay and also decreased the expression of several coactivators and downstream targets of Wnt signaling such as β-catenin, TCF7L2, p300, Bcl9l, c-Myc, vimentin, and cyclinD1 in SW620 colorectal cancer cells. Using isogenic HCT116p53+/+ and HCT116p53-/- colorectal cancer cells, it was found that compound 1-mediated downregulation of Wnt signaling is p53-independent. Interestingly, treatment with 1 upregulated the expression of HBP1 (a negative regulator of Wnt signaling) in these cells. The results obtained show for the first time that downregulation of Wnt signaling could be one of the molecular mechanisms by which plumbagin exerts its inhibitory effects in human colorectal cancer cells.

Topics: Apoptosis; beta Catenin; Cell Proliferation; Colorectal Neoplasms; Down-Regulation; Humans; Molecular Structure; Naphthoquinones; Signal Transduction; Tumor Suppressor Protein p53; Wnt Proteins; Wnt Signaling Pathway

We have synthesized, structurally characterized and examined cytotoxicity of novel plumbagin hydrazones against estrogen and progesterone receptor positive (ER+/PR+) MCF-7 and triple negative MDA-MB-231 breast cancer cell lines in order to evaluate the potential of these novel phytochemical analogs. Compounds were docked into the protein cavity of p50-subunit of NF-κB protein revealing better fit and better binding energies than the parent plumbagin compound. This was also reflected in their superior cytotoxicities which were found to be mediated by inhibition of NF-κB expression. These compounds can provide a starting point for the development of novel drug molecules against triple negative breast cancers.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; Hydrazones; MCF-7 Cells; Models, Molecular; Molecular Structure; Naphthoquinones; Structure-Activity Relationship

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

Plumbagin (PL, 5-hydroxy-2-methyl-1,4-naphthoquinone) is a herbal compound derived from medicinal plants of the Droseraceae, Plumbaginaceae, Dioncophyllaceae, and Ancistrocladaceae families. Reports have shown that PL exerts immunomodulatory activity and may be a novel drug candidate for immune-related disease therapy. However, its effects on dendritic cells (DCs), the most potent antigen-presenting cells (APCs), remain unclear. In this study, we demonstrate that PL inhibits the differentiation, maturation, and function of human monocyte-derived DCs. PL can also restrict the expression of Th1- and Th17-polarizing cytokines in mDC. In addition, PL suppresses DCs both in vitro and in vivo, as demonstrated by its effects on the mouse DC line DC2.4 and mice with experimental autoimmune encephalomyelitis (EAE), respectively. Notably, PL ameliorated the clinical symptoms of EAE, including central nervous system (CNS) inflammation and demyelination. Our results demonstrate the immune suppressive and anti-inflammatory properties of PL via its effects on DCs and suggest that PL could be a potential treatment for DC-related autoimmune and inflammatory diseases.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD; Apoptosis; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; Cytokines; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endocytosis; Female; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Naphthoquinones; Spinal Cord

Plumbagin is a plant naphtoquinone exerting anti-cancer properties including apoptotic cell death induction and generation of reactive oxygen species (ROS). The aim of this study was to elucidate parameters explaining the differential leukemia cell sensitivity towards this compound. Among several leukemia cell lines, U937 monocytic leukemia cells appeared more sensitive to plumbagin treatment in terms of cytotoxicity and level of apoptotic cell death compared to more resistant Raji Burkitt lymphoma cells. Moreover, U937 cells exhibited a ten-fold higher ROS production compared to Raji. Neither differential incorporation, nor efflux of plumbagin was detected. Pre-treatment with thiol-containing antioxidants prevented ROS production and subsequent induction of cell death by apoptosis whereas non-thiol-containing antioxidants remained ineffective in both cellular models. We conclude that the anticancer potential of plumbagin is driven by pro-oxidant activities related to the cellular thiolstat.

Topics: Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Cell Line, Tumor; Cell Survival; Glutathione; Humans; Leukemia; Naphthoquinones; Oxidation-Reduction; Reactive Oxygen Species; U937 Cells

Despite several phytochemical studies of Nepenthes gracilis Korth (Nepenthaceae), the biological activities of this pitcher plant remain to be explored.. This study evaluates the antifungal activity of N. gracilis extracts, isolates, and characterizes its bioactive compound and evaluates the cytotoxicity of the isolated compound.. Fresh leaves of N. gracilis were sequentially extracted. The fungistatic and fungicidal activities of the extracts were evaluated against six species of fungi of medical importance using a colorimetric broth microdilution method. The most active extract was fractionated by liquid-liquid partitioning and further purified by a preparative thin layer chromatography. Structural elucidation was carried out using FT-IR, GC-MS, and NMR. Cytotoxicity testing against rhesus monkey kidney epithelial cells (LLC-MK2) was assessed by a neutral red uptake (NRU) assay.. The hexane extract, which showed the lowest minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), both at 20 μg/mL against Candida albicans, Issatchenkia orientalis, and Trichophyton mentagrophytes, was subjected to bioactivity-guided fractionation. The isolated compound exhibited potent activity with the MIC values ranging from 2 to 31 μg/mL against all the fungi. The active compound was identified as plumbagin (5-hydroxy-2-methyl-naphthalene-1,4-dione). The 50% cytotoxicity concentration (CC50) of plumbagin was 0.60 μg/mL.. The selectivity indices of plumbagin against all the fungi were less than 1.0, indicating that plumbagin is more toxic to mammalian than fungal cells. This study provides information on the antifungal properties of N. gracilis leaf extracts, as well as the antifungal and cytotoxicity properties of plumbagin.

Topics: Animals; Antifungal Agents; Cell Line; Chromatography, Thin Layer; Colorimetry; Fungi; Macaca mulatta; Magnoliopsida; Microbial Sensitivity Tests; Naphthoquinones; Plant Extracts; Plant Leaves

In the present investigation, the effective root compound of plumbagin of Plumbago zeylanica (Plumbaginaceae) was evaluated for chemical constituent and antimalarial effect against the fourth instar larvae of Anopheles stephensi Liston (Diptera). In the chromatographic analyses of root compound with Rf value of 0.788 and NMR analyses also revealed that the effective compound contain naphthoquinone plumbagin were identified as the major chemical constituent. Larval mortality was observed after 3 h of exposure period. The plumbagin compound showed remarkable larvicidal activity against A. stephensi (LC50 32.65 and LC9072.27 ppm). Histopathological effects of compound was observed in the treated larvae. Based on the results, the plumbagin compound of P. zeylanica can be considered as a new source of natural larvicide for the control of malarial vector.

Topics: Animals; Anopheles; Insecticides; Larva; Naphthoquinones; Plant Extracts; Plant Roots; Plumbaginaceae

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

Despite increased use of early detection methods and more aggressive treatment strategies, the worldwide incidence of colorectal cancer is still on the rise. Consequently, it remains urgent to identify novel agents with enhanced efficacy in prevention and/or therapeutic protocols. Our studies focused on the use of Plumbagin, a natural phytochemical that showed promising results against other tumor types, to determine its effectiveness in blocking the proliferation and survival of colon cancer cells in experimental protocols mimicking the environment in primary tumors (attached culture conditions) and in circulating tumor cells (unattached conditions). Under both experimental settings, exposure of HCT116 cells to Plumbagin concentrations in the low micromolar range resulted in cell cycle arrest at the G1 phase, apoptosis via the mitochondrial cell death pathway, and increased production of reactive oxygen species. The cell cycle effects were more noticeable in attached cells, whereas the induction of cell death was more evident in unattached cells. These effects were consistent with the nature and the magnitude of the alterations induced by Plumbagin on the expression levels of a set of proteins known to play key roles in the regulation of cell cycle dynamics, apoptosis mechanisms and cell proliferation. In light of its previously reported lack of toxicity on normal colon cells and the striking anti-survival effect on colon cancer cells observed in our study, Plumbagin should be considered a promising drug for the treatment of colon cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle Checkpoints; Cell Proliferation; Cell Survival; Colonic Neoplasms; HCT116 Cells; Humans; Mitochondria; Naphthoquinones; Reactive Oxygen Species

Plumbagin is a nutraceutical with potent anti-cancer activity. However, the therapeutic efficacy of plumbagin is overshadowed by the lack of sensitivity and selectivity towards cancer cells. The present study evaluated the use of nano-biotechnological intervention to cage plumbagin in silver nanoparticles for selective targeting of its biological effects towards cancerous cells. Caging of plumbagin in silver nanoparticles imparted selectivity and sensitivity to plumbagin for selective killing of cancer cells by altering the redox signalling events in the cancer cells. The selectivity and sensitivity of plumbagin towards cancer cells was due to the cumulative expression of the properties of plumbagin and nanoparticles which specifically affected the differential cancer cell microenvironment by altering the pyruvate kinase activity that regulates the ROS challenge in cancerous cells. The positive surface charge of plumbagin caged silver nanoparticles (PCSN) aids in getting them targeted towards anionic cancerous cells due to the exposed terminal carboxyl group of sialic acid residues. Furthermore, we observed that the effective concentration of the drug required to induce apoptosis was brought down to 50% upon caging of plumbagin on silver nanoparticles. We observed no such effect with the individual compound alone. The results indicated that the physico-chemical and biochemical properties of plumbagin significantly changed after conjugation with nanomaterials that facilitated "adding-in" therapeutical values to plumbagin which would otherwise be overshadowed by its lack of sensitivity and selectivity towards cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Drug Delivery Systems; Humans; Metal Nanoparticles; Naphthoquinones; Silver

A novel Plumbagin-Isoniazid Analog (PLIHZ) and its β-cyclodextrin inclusion complex (PLIHZCD) is prepared, characterized and evaluated for antitubercular activity under low and high iron conditions. PLIHZCD inclusion complex was characterized by Fourier Transform Infra-Red (FTIR), Differential Scanning Calorimetry (DSC), Powder X-ray Diffraction Studies (PXRD), (1)H NMR Studies and Scanning Electron Microscopic (SEM) analysis. The orientation and interaction of PLIHZ and CD was studied by molecular docking. PLIHZCD exhibited superior activity (MIC of 4 μg/ml) than PLIHZ and PL under 7H9 medium conditions. The standard anti-tubercular compound INH exhibited MIC values of 0.125 and 32 μg/ml under high and low iron conditions, whereas the conjugate PLIHZ exhibited MIC values of 0.5 and 2.0 μg/ml under high and low iron (corresponding to isoniazid resistant condition) indicating the advantage of combining plumbagin with INH overcoming resistance. The cyclodextrin conjugate offers improved aqueous solubility and thermal stability which are advantages in the treatment protocol.

Topics: Antitubercular Agents; beta-Cyclodextrins; Calorimetry, Differential Scanning; Drug Stability; Isoniazid; Microscopy, Electron, Scanning; Molecular Conformation; Molecular Dynamics Simulation; Mycobacterium tuberculosis; Naphthoquinones; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Oxidative stress plays a pivotal role in the impaired endothelial function occurring in vascular diseases. Antioxidant strategies induce a clinical advantage in patients with endothelial dysfunction and atherosclerosis and protect from oxidative damage, but the underlying molecular mechanisms have been poorly evaluated. The aim of this study was to analyze the effects and mechanisms of action of antioxidant regimens on endothelial function.. Antioxidant efficacy of N-acetylcysteine, ascorbic acid and propionyl-L-carnitine was evaluated in serum-deprived and TNF-α-stimulated human umbilical vein endothelial cells in vitro. Cell adhesion molecule (CAM) expression was evaluated by blot and real-time PCR, and inflammatory cytokine secretion was evaluated by ELISA; leukocyte adhesion and reactive oxygen species assays and NADPH oxidase 4 isoform (Nox4) expression analyses by blots were also performed. Antioxidant pretreatment restored serum-deprived and TNF-α-induced impaired mitochondrial β-oxidation by reducing flavin adenine dinucleotide level and counteracting increased CAM and Nox4 expression, leukocyte adhesion and inflammatory cytokine secretion. Specific inhibition by plumbagin and siNox4 prevented TNF-α- and serum deprivation-induced detrimental effects, confirming that endothelial oxidative stress and inflammation were Nox4 dependent.. Our findings documented Nox4 as a main actor in oxidative stress-induced endothelial dysfunction and further clarify the molecular basis of antioxidant treatment efficacy.

Topics: Acetylcysteine; Antioxidants; Ascorbic Acid; Carnitine; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Coculture Techniques; Cytokines; Cytoprotection; Enzyme Inhibitors; Human Umbilical Vein Endothelial Cells; Humans; Leukocytes; Mitochondria; NADPH Oxidase 4; NADPH Oxidases; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; RNA Interference; Serum; Transfection; Tumor Necrosis Factor-alpha

The medicinal plant Plumbago contains a very potent secondary metabolite, plumbagin having many therapeutic properties. Callus culture was induced using explants, leaf, stem and shoot apex, from P. auriculata. Murashige and Skoog media fortified with various growth hormones like NAA, IAA, IBA and 2, 4-D individually and in various combinations were checked for callus induction. Among the growth hormones used, 1 mg/L 2, 4-D showed best callusing. The hormonal combinations of 1 mg/L IAA and 1.5 mg/L NAA in the media exhibited best callus induction using stem internode as an explant. Plumbagin content from root, stem, leaf and callus was analyzed by using thin layer chromatographic technique. The callus derived from stem showed comparable plumbagin content to the in vivo plant parts. Quantitative spectrophotometric analysis of plumbagin from plant samples and callus indicated that plumbagin content was maximum in roots which was followed by callus, stem and leaf samples respectively. Generation of in vitro sources for p!umbagin, for therapeutic applications will serve as a continuous supply and will contribute to preserve the natural plant recourses.

Topics: Chromatography, Thin Layer; Colorimetry; Cytokinins; Indoleacetic Acids; Naphthoquinones; Organ Specificity; Organoids; Plant Cells; Plant Leaves; Plant Roots; Plant Shoots; Plant Stems; Plants, Medicinal; Plumbaginaceae; Tissue Culture Techniques

Benjakul, a Thai traditional herbal preparation, comnprises five plants: Piper chaba, Piper sarmentosum, Piper interruptum, Plumbago indica, and Zingiber officinale. It has widely been used to treat cancer patients in folk medicine in Thailand. Benjakul extract, and its isolated compounds should be investigated for cytotoxic activity and analysis isolated compounds from chemical fingerprinting.. To study cytotoxicity ofBenjakul extract and its isolatedpure compounds against human small cell lung cancer cell line (NCI-HI 688) and in normal human lungfibroblast cell line (MRC-5) and analysis the content ofisolated compounds for quality control of Benjakul extract.. Bioassay-guided fractionation was used for isolated active compounds from ethanolic extract of Benjakul. Cytotoxic activity was carried using the SRB assay. HPLC method was applied to analyze six isolated compound contentfrom Benjakul extract.. The ethanolic extract ofBenjakul showed cytotoxicity against NCI-H1688 with IC50 value = 36.15±4.35 μg/ml. Hexane fraction as semi-separation by VLC showed the best cytotoxic activity (21.1 7±7.42 μg/ml). Six isolated compounds were identified as myristicin, plumbagin, methyl piperate, 6-shogaol, 6-gingerol and piperine. Plumbagin exhibited the highest cytotoxic activity and 6-shogaol was the second most effective cytotoxic constituent (IC50 values = 1.41±0.01 and 6.45±0.19 μg/ml, respectively). Piperine showed the highest content in both ofHPLC analysis and column chromatography separation.. Benjakul extract exhibited cytotoxicity against NCI-HI 688. Plumbagin and 6-shogaol are bioactive markers for cytotoxicity against this small cell lung cancer cell line. Chromatographic fingerprinting can be used to analyze six cytotoxic compounds isolatedfrom the ethanolic extract ofBenjakul.

Topics: Alkaloids; Benzodioxoles; Catechols; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Screening Assays, Antitumor; Ethanol; Fatty Alcohols; Humans; Lung Neoplasms; Medicine, Traditional; Naphthoquinones; Piper; Piperidines; Plant Extracts; Plumbaginaceae; Polyunsaturated Alkamides; Small Cell Lung Carcinoma; Thailand; Zingiber officinale

Staphylococcus aureus is the most common infectious agent involved in the development of skin infections that are associated with antibiotic resistance, such as burn wounds. As drug resistance is a growing problem it is essential to establish novel antimicrobials. Currently, antibiotic resistance in bacteria is successfully controlled by multi-drug therapies. Here we demonstrate that secondary metabolites present in the extract obtained from Drosera binata in vitro cultures are effective antibacterial agents against S. aureus grown in planktonic culture and in biofilm. Moreover, this is the first report demonstrating the synergistic interaction between the D. binata extract and silver nanoparticles (AgNPs), which results in the spectacular enhancement of the observed bactericidal activity, while having no cytotoxic effects on human keratinocytes. Simultaneous use of these two agents in significantly reduced quantities produces the same effect, i.e. by killing 99.9% of bacteria in inoculum or eradicating the staphylococcal biofilm, as higher amounts of the agents used individually. Our data indicates that combining AgNPs with either the D. binata extract or with its pure compound (3-chloroplumbagin) may provide a safe and highly effective alternative to commonly used antibiotics, which are ineffective towards the antibiotic-resistant S. aureus.

Topics: Anti-Bacterial Agents; Biofilms; Burns; Cells, Cultured; Drosera; Drug Resistance, Multiple, Bacterial; Humans; Keratinocytes; Metal Nanoparticles; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Naphthoquinones; Plant Extracts; Silver; Silver Compounds; Wound Infection

Here we show that a series of inhibitors, constructed from plumbagin conjugated to a phenyl thioglucoside via an alkyl chain of variable length, are bound in solution-favoured ligand conformations to a mycothiol biosynthetic enzyme MshB, a GlcNAc-Ins deacetylase. The kinetic studies of this ligand series show that MshB is more strongly inhibited as a function of increasing alkyl chain length. While docking studies yielded highest ranked conformations in which the ligands extended along the catalytic site, these conformations produced free energy values prone to large errors and which were inconsistent with experimental kinetic measurements. Solution-favoured conformations of the inhibitors feature a preference for intramolecular aromatic association that results in curled conformations. Free energy perturbation calculations of MshB bound to the inhibitors in the preconfigured solution-favoured curled conformations gave the same binding pattern observed in the kinetic experiments. On investigation of these conformations lodged in the catalytic domain, we found that the selective feature determining their relative binding strength was the result of an optimisation of the dispersion interactions between the ligand aromatic groups phenyl and plumbagin, and the enzyme aromatic groups His144 and Tyr142 respectively. These results show that rather than deform the preferred folded ligand solution conformation, such that the hydrophobic C-2 acyl chain is linearly projected into a buried hydrophobic rich binding cavity adjacent to the active site, MshB binds preconfigured solution inhibitor curled conformations with a preference for aromatic association.

Topics: Amidohydrolases; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Cysteine; Enzyme Inhibitors; Glycopeptides; Inositol; Kinetics; Ligands; Molecular Conformation; Molecular Docking Simulation; Mycobacterium tuberculosis; Naphthoquinones; Protein Structure, Secondary; Superoxides; Thermodynamics; Thioglucosides

A biomimetic synthesis of zeylanone and zeylanone epoxide, which are natural dimeric naphthoquinones, has been accomplished starting from plumbagin, a natural monomeric naphthoquinone. The key features of our synthesis are cascade intermolecular and intramolecular Michael reactions, followed by epoxidation of the resultant hydroquinone with molecular oxygen.

Topics: Biomimetics; Dimerization; Epoxy Compounds; Hydroquinones; Molecular Structure; Naphthoquinones; Vitamin K 3

Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity.

Topics: Animals; Astrocytes; Carrier Proteins; Cell Death; Cell Survival; Dose-Response Relationship, Drug; Genes, Reporter; Glutamic Acid; Hep G2 Cells; Humans; Microfilament Proteins; Naphthoquinones; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Primary Cell Culture; Proteolysis; Rats; Rats, Sprague-Dawley

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

Here we report that activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in both primary cultured human colon cancer cells and cell lines. Knocking-down of AMPKα by the target shRNA significantly inhibits plumbagin-induced cytotoxicity in cultured colon cancer cells, while forced activation of AMPK by introducing a constitutively active AMPK (CA-AMPK), or by the AMPK activator, inhibits HT-29 colon cancer cell growth. Our Western-blots and immunoprecipitation (IP) results demonstrate that plumbagin induces AMPK/Apoptosis signal regulating kinase 1 (ASK1)/TNF receptor-associated factor 2 (TRAF2) association to activate pro-apoptotic c-Jun N-terminal kinases (JNK)-p53 signal axis. Further, after plumbagin treatment, activated AMPK directly phosphorylates Raptor to inhibit mTOR complex 1 (mTORC1) activation and Bcl-2 expression in colon cancer cells. Finally, we found that exogenously-added short-chain ceramide (C6) enhances plumbagin-induced AMPK activation and facilitates cell apoptosis and growth inhibition. Our results suggest that AMPK might be the key mediator of plumbagin's anti-tumor activity.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Naphthoquinones; Phosphorylation; Primary Cell Culture; Reactive Oxygen Species; Signal Transduction

To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer cell lines, and investigate the underlying mechanisms.. Human non-small cell lung cancer cell lines A549, H292 and H460 were treated with various concentrations of plumbagin. Cell proliferation rates was determined using both cell counting kit-8 (CCK-8) and clonogenic assays. Apoptosis was detected by annexin V/propidium iodide double-labeled flow cytometry and TUNEL assay. The levels of reactive oxygen species (ROS) were detected by flow cytometry. Activity of NF-κB was examined by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of both NF-κB regulated apoptotic-related gene and activation of p65 and IκBκ.. Plumbagin dose-dependently inhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 μmol/L, 7.3 μmol/L, and 6.1 μmol/L for 12 hours, respectively. The compound concentration-dependently induced apoptosis of the three cell lines. Treatment with plumbagin increased the intracellular level of ROS, and inhibited the activation of NK-κB. In addition to inhibition of NF-κB/p65 nuclear translocation, the compound also suppressed the degradation of IκBκ. ROS scavenger NAC highly reversed the effect of plumbagin on apoptosis and inactivation of NK-κB in H460 cell line. Treatment with plumbagin also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl-2, upregulated the expression of Bax, Bak, and CytC.. Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells through an NF-κB-regulated mitochondrial-mediated pathway, involving activation of ROS.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Electrophoretic Mobility Shift Assay; Humans; Luciferases; Lung Neoplasms; Naphthoquinones; NF-kappa B; Plumbaginaceae; Protein Transport; Superoxides; Tumor Cells, Cultured

Plumbagin (1) is a naphthoquinone constituent of plants that have been used in traditional systems of medicine since ancient times. In the present study, the role of 1 was examined on the amelioration of ulcerative colitis, an inflammatory bowel disease that is not curable currently. Plumbagin was tested at a dose of 6-10 mg/kg body weight in acute and chronic disease models. Diseased mice receiving 1 at 8-10 mg/kg demonstrated a significant suppression of disease symptoms in both models. However, body weight loss was not restored in either of the models. Levels of proinflammatory cytokines (TNF-α, IFN-γ, and IL-17) were reduced significantly by 1 in mice suffering from chronic disease, while cytokine levels remained unaffected in mice with acute disease. However, the percentage of inflammatory (CD14+/CD16+) monocytes present in peripheral blood was significantly reduced by >3-fold (p < 0.05) in treatment groups relative to controls in the acute model. Histological evaluations exhibited the restoration of goblet cells, crypts, and the submucosa along with a significant reduction in monocyte aggregation in colon sections from mice receiving treatment with 1. Restoration in colon size was also observed in the treatment groups.

Topics: Animals; Colitis, Ulcerative; Colon; Cytokines; Disease Models, Animal; Interleukin-17; Male; Mice; Molecular Structure; Naphthoquinones; Tumor Necrosis Factor-alpha

Plumbagin is a major active constituent of Plumbago indica L. (Plumbaginaceae). It possesses various pharmacological activities that have been shown to assist in the treatment of various diseases.. This work is focused on increasing the production of plumbagin in P. indica root cultures using low doses of gamma ray irradiation as an elicitor.. The effect of low doses of gamma ray irradiation (0, 5, 10, 15, 20, 25 Gy) and ages of the root cultures (0, 5, 10, 15, 20 days) for elicitation of plumbagin production was determined. The stability of the elicited root cultures to produce plumbagin was also determined during three cycles of subculture.. Treatment of the root cultures with a low dose of gamma ray at 20 Gy gave the highest level of plumbagin production (1.04 mg/g DW) when compared to all other treated groups. The appropriate age of the root cultures for maximum production of plumbagin was found to be 10 days. However, treatment of 5-day-old root cultures resulted in a significant increase of dried root biomass that also had a high plumbagin production. Based on the total biomass per culture flask, the amounts of plumbagin produced by the 5- and 10-day-old treated roots were 0.59 and 0.37 mg/250 mL flask, respectively, which were 4.2- and 2.6-fold higher than the level in the control. Subculturing the root cultures until the third generation still showed an increase in plumbagin production without any effects on their growth.

Topics: Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Gamma Rays; Naphthoquinones; Plant Roots; Plumbaginaceae; Time Factors

The assembly of FtsZ plays a central role in construction of the cytokinetic Z-ring that orchestrates bacterial cell division. A naturally occurring naphthoquinone, plumbagin, is known to exhibit antibacterial properties against several types of bacteria. In this study, plumbagin was found to perturb formation of the Z-ring in Bacillus subtilis 168 cells and to cause elongation of these cells without an apparent effect on nucleoid segregation, indicating that it may inhibit FtsZ assembly. Furthermore, it bound to purified B. subtilis FtsZ (BsFtsZ) with a dissociation constant of 20.7 ± 5.6 μM, and inhibited the assembly and GTPase activity of BsFtsZ in vitro. Interestingly, plumbagin did not inhibit either the assembly or GTPase activity of Escherichia coli FtsZ (EcFtsZ) in vitro. Using docking analysis, a putative plumbagin-binding site on BsFtsZ was identified, and the analysis indicated that hydrophobic interactions and hydrogen bonds predominate. Based on the in silico analysis, two variants of BsFtsZ, namely D199A and V307R, were constructed to explore the binding interaction of plumbagin and BsFtsZ. The effects of plumbagin on the assembly and GTPase activity of the variant BsFtsZ proteins in vitro indicated that the residues D199 and V307 may be involved in the binding of plumbagin to BsFtsZ. The results suggest that plumbagin inhibits bacterial proliferation by inhibiting the assembly of FtsZ, and provide insight into the binding site of plumbagin on BsFtsZ, which may help in the design of potent FtsZ-targeted antibacterial agents.

Topics: Anti-Bacterial Agents; Aspartic Acid; Bacillus subtilis; Bacterial Load; Bacterial Proteins; Binding Sites; Cytokinesis; Cytoskeletal Proteins; Escherichia coli; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Mutation; Naphthoquinones; Protein Binding; Recombinant Proteins; Species Specificity; Valine

The transcriptional regulator QsrR is converted into a genetically encoded fluorescent probe capable of ratiometric monitoring of quinones in living cells with high sensitivity and selectivity.

Topics: Fluorescent Dyes; Gene Expression Regulation; HeLa Cells; Humans; Limit of Detection; Luminescent Proteins; Molecular Imaging; Naphthoquinones; Quinones

Antimicrobial lethality is promoted by reactive oxygen species (ROS), such as superoxide, peroxide, and hydroxyl radical. Pretreatment with subinhibitory concentrations of plumbagin or paraquat, metabolic generators of superoxide, paradoxically reduced killing for oxolinic acid, kanamycin, and ampicillin. These pretreatments also reduced an oxolinic acid-mediated ROS surge. Defects in SoxS MarA or AcrB eliminated plumbagin- and paraquat-mediated MIC increases but maintained protection from killing. Thus, superoxide has both protective and detrimental roles in response to antimicrobial stress.

Topics: Ampicillin; Anti-Bacterial Agents; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance, Bacterial; Escherichia coli K12; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Kanamycin; Microbial Sensitivity Tests; Multidrug Resistance-Associated Proteins; Naphthoquinones; Oxolinic Acid; Paraquat; Superoxides; Trans-Activators

Plumbagin, a quinonoid constituent isolated from the root of Plumbago zeylanica L., has been proven to possess anti-tumor activity both in vitro and in vivo. However, its anti-tumor properties for human tongue carcinoma have not been reported. This study aimed to investigate the inhibitory effect and the underlying mechanism of plumbagin on the growth of human tongue carcinoma cells.. Cell proliferation ability was detected by EdU incorporation assay and colony formation assay. Cell-cycle distribution was determined by flow cytometric analysis using propidium iodide (PI) staining. Cellular apoptosis was then evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Western blotting was applied to assay the expression of Bax and Bcl-2.. Plumbagin inhibited the growth and proliferation of Tca8113 cells in vitro in a concentration- and time-dependent manner. The cell cycles of plumbagin-treated Tca8113 cells were arrested at the G2/M phase. Cells treated with plumbagin presented the characteristic morphological changes of apoptosis. The ratio of Bax/Bcl-2 was raised by plumbagin in a concentration-dependent manner.. These results indicate that plumbagin induces the apoptosis of Tca8113 cells through mitochondria-mediated pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cell Survival; Deoxyuridine; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Humans; Mitochondria; Mitosis; Naphthoquinones; Signal Transduction; Time Factors; Tongue Neoplasms; Tumor Stem Cell Assay

NADPH oxidase 4 (Nox4) is reported to be the major source of reactive oxygen species (ROS) in the kidneys during the early stages of diabetic nephropathy. It has been shown to mediate TGFβ1-induced differentiation of cardiac fibroblasts into myofibroblasts. Despite TGFβ1 being recognised as a mediator of renal fibrosis and functional decline role in diabetic nephropathy, the renal interaction between Nox 4 and TGFβ1 is not well characterised. The aim of this study was to investigate the role of Nox4 inhibition on TGFβ1-induced fibrotic responses in proximal tubular cells and in a mouse model of diabetic nephropathy. Immortalised human proximal tubular cells (HK2) were incubated with TGFβ1 ± plumbagin (an inhibitor of Nox4) or specific Nox4 siRNA. Collagen IV and fibronectin mRNA and protein expression were measured. Streptozotocin (STZ) induced diabetic C57BL/6J mice were administered plumbagin (2 mg/kg/day) or vehicle (DMSO; 50 µl/mouse) for 24 weeks. Metabolic, physiological and histological markers of nephropathy were determined. TGFβ1 increased Nox4 mRNA expression and plumbagin and Nox4 siRNA significantly inhibited TGF-β1 induced fibronectin and collagen IV expression in human HK2 cells. STZ-induced diabetic C57BL/6J mice developed physiological features of diabetic nephropathy at 24 weeks, which were reversed with concomitant plumbagin treatment. Histologically, plumbagin ameliorated diabetes induced upregulation of extracellular matrix protein expression compared to control. This study demonstrates that plumbagin ameliorates the development of diabetic nephropathy through pathways that include Nox4 signalling.

Topics: Animals; Base Sequence; Cell Line, Transformed; Collagen Type IV; Diabetic Nephropathies; DNA Primers; Fibronectins; Glucose; Humans; Mice; Mice, Inbred C57BL; NADPH Oxidases; Naphthoquinones; Real-Time Polymerase Chain Reaction; RNA, Messenger; Streptozocin; Superoxide Dismutase; Transforming Growth Factor beta1

The water maceration and methanolic extract of the stem barks of Diospyros bipindensis, which is a medicinal plant used in Cameroon by Baka pygmies, revealed a complex high-performance liquid chromatography (HPLC) profile primarily composed of coumarin and naphthoquinone glycosides. The methanolic and apolar extracts also exhibited significant antifungal activity on a TLC bioautography assay against Candida albicans. HPLC-microfractionation in 96-well plates combined with bioautography enabled the rapid localization of the antifungal compound that was identified by HPLC-PDA and HPLC-MS analysis as plumbagin. These on-line structural information were also used to dereplicate four known compounds. The isolation of the polar constituents from the methanolic extract enabled the identification of eleven other compounds also present in the traditional preparation, nine of which are reported for the first time. The structures of those compounds were elucidated by UV, NMR and HR-MS analysis.

Topics: Antifungal Agents; Cameroon; Candida albicans; Chromatography, High Pressure Liquid; Coumarins; Diospyros; Glycosides; Growth Disorders; Medicine, African Traditional; Methanol; Microbial Sensitivity Tests; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Phenols; Plant Extracts

MarR is the dedicated autorepressor of the marRAB operon found in seven genera of the Enterobacteraceae. The MarA transcriptional regulator directly activates numerous genes involved in multidrug resistance and other environmental responses. MarR is inactivated by certain phenolic ligands, such as salicylate, by an unknown mechanism. Our recent work has shown that several amino acid residues of Escherichia coli MarR affecting ligand binding are located between the dimerization and DNA-binding domains. To further characterize the ligand-binding region of MarR, we have now examined 7 point mutants generated by random mutagenesis and 11 site-directed alanine replacement mutants for inactivation by three ligands: salicylate, 2,4-dinitrophenol, and plumbagin. Inactivation of MarR was quantitated in intact cells by loss of MarR-mediated repression of a chromosomal mar-lacZ transcriptional fusion. The results showed that most of the residues important for ligand effectiveness lay in the α1 and α2 helices of MarR, between the putative DNA-binding domain and the dimerization domain of MarR, reinforcing our earlier findings. Moreover, the three ligands had different, but overlapping, sets of residues impacting their effects on MarR.

Topics: 2,4-Dinitrophenol; Amino Acids; Escherichia coli; Escherichia coli Proteins; Gene Silencing; Ligands; Models, Molecular; Mutagenesis; Mutation; Naphthoquinones; Protein Binding; Protein Structure, Tertiary; Repressor Proteins; Salicylates

Phospholipid and Tween(®) 80 mixed micelles were investigated as injectable nanocarriers for the natural anticancer compound, plumbagin (PBG), with the aim to improve anticancer efficiency. PBG-loaded mixed micelles were fabricated by self-assembly; composition being optimized using 3(2) factorial design.. Optimized mixed micelles were spherical and 46 nm in size. Zeta potential, drug loading and encapsulation efficiency were 5.04 mV, 91.21 and 98.38% respectively. Micelles demonstrated sustained release of PBG. Micelles caused a 2.1-fold enhancement in vitro antitumor activity of PBG towards MCF-7 cells. Micelles proved safe for intravenous injection as PBG was stable at high pH; micelle size and encapsulation efficiency were retained upon dilution.. Developed mixed micelles proved potential nanocarriers for PBG in cancer chemotherapy.

Topics: Antineoplastic Agents, Phytogenic; Blood Proteins; Breast Neoplasms; Cell Survival; Chemistry, Pharmaceutical; Delayed-Action Preparations; Drug Carriers; Drug Stability; Female; Hemolysis; Humans; Kinetics; MCF-7 Cells; Micelles; Nanoparticles; Nanotechnology; Naphthoquinones; Particle Size; Phospholipids; Polysorbates; Protein Binding; Solubility; Technology, Pharmaceutical

To investigate the effect of plumbagin on the expression of TNF-alpha and PDGF-BB in human hepatic stellate cells (HSC-LX2) activated by Leptin.. HSC-LX2 were cultured in vitro and stimulated by Leptin for 24 hours then treated with different concentrations of plumbagin for 24 hours, the expressions of TNF-alpha mRNA and PDGF-BB mRNA were determined by Realtime quantitative PCR, the protein expressions of TNF-alpha and PDGF-BB were determined-by Western blotting.. The expressions of TNF-alpha mRNA and PDGF-BB mRNA of treatment groups were significantly reduced, especially in high dose group (P < 0.01), and Western blotting analyses revealed similar trends in protein expression.. Plumbagin may prevent the formation of hepatic fibrosis and its mechanism may be related to decreasing the level of mRNA of TNF-alpha and PDGF-BB and the protein of PDGF-BB.

Topics: Becaplermin; Cells, Cultured; Gene Expression Regulation; Hepatic Stellate Cells; Humans; Leptin; Liver Cirrhosis; Naphthoquinones; Plumbaginaceae; Polymerase Chain Reaction; Proto-Oncogene Proteins c-sis; RNA, Messenger; Tumor Necrosis Factor-alpha

Human amniotic fluid stem cells (AFSC) with multilineage differentiation potential are novel source for cell therapy. However, in vitro expansion leads to senescence affecting differentiation and proliferative capacities. Reactive oxygen species (ROS) have been involved in the regulation of stem cell pluripotency, proliferation, and differentiation. Redox-regulated signal transduction is coordinated by spatially controlled production of ROS within subcellular compartments. NAD(P)H oxidase family, in particular Nox4, has been known to produce ROS in the nucleus; however, the mechanisms and the meaning of this function remain largely unknown. In the present study, we show that Nox4 nuclear expression (nNox4) increases during culture passages up to cell cycle arrest and the serum starvation causes the same effect. With the decrease of Nox4 activity, obtained with plumbagin, a decline of nuclear ROS production and of DNA damage occurs. Moreover, plumbagin exposure reduces the binding between nNox4 and nucleoskeleton components, as Matrin 3. The same effect was observed also for the binding with phospho-ERK, although nuclear ERK and P-ERK are unchanged. Taken together, we suggest that nNox4 regulation may have important pathophysiologic effects in stem cell proliferation through modulation of nuclear signaling and DNA damage.

Topics: Adult; Amniotic Fluid; Cell Nucleus; Cell Proliferation; DNA Damage; Female; Fluorescent Antibody Technique; Humans; NADPH Oxidase 4; NADPH Oxidases; Naphthoquinones; Protein Transport; Reactive Oxygen Species; Serum; Stem Cells; Subcellular Fractions

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

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti-angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti-proliferative and anti-angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO-1 and cisplatin resistant, BRCA2 proficient PEO-4 ovarian cancer cells. Both PEO-1 and PEO-4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF-A and Glut-1 in PEO-1 and PEO-4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR-5 cells. Plumbagin challenge also restricts the VEGF induced pro-angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR-5 tumor-bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti-cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; BRCA2 Protein; Calcium; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cisplatin; Endothelial Cells; Female; Glucose Transporter Type 1; Humans; Ki-67 Antigen; Mice; Mice, SCID; Naphthoquinones; Neovascularization, Pathologic; Ovarian Neoplasms; Platelet Endothelial Cell Adhesion Molecule-1; Random Allocation; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Hormonal ablation is the standard treatment for disseminated androgen-dependent prostate cancer. Although tumor growth is controlled at first, the tumor invariably recurs in the form of castration-resistant prostate cancer. This study assessed the efficacy of a new therapeutic strategy that combines plumbagin, a naturally occurring naphthoquinone, with androgen ablation.. Viewing microscopy chambers were placed in the dorsal skinfold of mice. Syngeneic prostate tissue was grafted within the chambers and allowed to vascularize. H2B-GFP/PTEN-P2 prostate cancer cells were co-implanted on top of the grafted prostate tissue. Androgen ablation was achieved using surgical castration. Intact and castrated mice were administered plumbagin or sham treatment. Tumor growth, mitosis and apoptosis were monitored in real-time using fluorescent Intra-Vital Microscopy. The mechanism of action of plumbagin was explored using human and mouse prostate cancer cells.. Whereas both plumbagin and castration alone impeded tumor growth, only the combination of plumbagin and castration caused profound tumor regression in vivo, mostly due to increased apoptosis of the tumor cells. The cytotoxicity of plumbagin was not affected by androgens in vitro, suggesting that microenvironmental factors not present in culture play a crucial role in the combination effect. Plumbagin-induced cell death was mediated, at least in part, by activation of ERK and was due to generation of reactive oxygen species, because it was abolished by the anti-oxidant N-acetyl-L-cysteine.. Androgen deprivation in combination with plumbagin may provide a significant improvement over androgen deprivation alone and deserves further evaluation.

Topics: Androgens; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; G2 Phase Cell Cycle Checkpoints; Green Fluorescent Proteins; Humans; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Neoplasm Transplantation; Orchiectomy; Reactive Oxygen Species; Skin

The anthelmintic effects of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) and praziquantel (PZQ) against adult Schistosoma mansoni in vitro were compared by estimating the relative motility (RM) values, survival indices (SI) and alterations of the tegument of flukes incubated in M-199 medium containing 1, 10 and 100 μg/ml of the drugs, at 1, 3, 6, 12 and 24 h. For the parasites incubated in 10 μg/ml, the RM values of the PB-treated group decreased at a more rapid rate than the PZQ-treated group. When incubated in 100 μg/ml all PB-treated flukes appeared immobile from 1 to 24 h when 91-100% died, while in the PZQ-treated group RM values were higher than that of PB and most flukes were still alive at 1-12 h, and at 24 h only 21% of flukes were killed. Furthermore, male parasites were more affected by PZQ than females as their RM values were significantly less than that of females at all doses. While in PB treatment, males and females showed less difference in response to the drug as their RM values were closer than those treated with PZQ. When observed by SEM, the tegument of untreated S. mansoni displayed no alteration, while in PB treated parasites it exhibited swelling, blebbing, loss of spines, disruption of tubercles and ridges, leading to erosion and lesion, exposure of the basal lamina, and sloughing of the tegument. PZQ induced similar tegumental changes as those observed in PB treatment but at longer incubation time and higher doses. These data indicated that PB had more anthelmintic effect on both sexes of adult S. mansoni than PZQ.

Topics: Animals; Anthelmintics; Biomphalaria; Female; Inhibitory Concentration 50; Male; Mice; Microscopy, Electron, Scanning; Naphthoquinones; Praziquantel; Schistosoma mansoni; Sex Factors

A phytochemical investigation of the stem bark of Diospyros cuneata (Ebenaceae) together with in vitro cytotoxic evaluation of the pure compounds in four human cancer cell lines KB, Hep-2, HeLa and SiHa led to the isolation of plumbagin, elliptinone, lupeol, atraric acid methyl ester, maritinone, betulin and betulinaldehyde. The structural determination of the compounds was established by nuclear magnetic resonance, electron ionisation mass spectrometric analysis as well as comparison with data from the literature. Plumbagin exhibited a potent cytotoxic activity (CC50 = 3.56 µg mL(-1)) against KB cell lines whereas maritinone displayed cytotoxic activity against Hep-2 (CC50 = 17.30 µg mL(-1)), cervical cancer (CC50 = 21.10 µg mL(-1)) and the KB cell lines (CC50 = 20.30 µg mL(-1)). This is the first report on a phytochemical and biological evaluation of D. cuneata.

Topics: Cell Line, Tumor; Cell Survival; Diospyros; Humans; Naphthoquinones; Pentacyclic Triterpenes; Plant Bark; Plant Extracts; Plant Stems; Triterpenes

This paper presents a comparative study of anatomy of leaves, stems and roots of three species of Plumbago, namely P. auriculata Lam., P. indica L. and P. zeylanica L. by light microscopy. The paper also provides qualitative and quantitative analysis of the naphthoquinone, plumbagin-a major constituent present in these species-using UPLC-UV. Microscopic examinations revealed the presence of distinctive differences in the anatomical features of the leaf, stem and root of the three species, and these can thus be used for identification and authentication of these species. UPLC-UV analysis showed the highest concentration of plumbagin in the roots of P. zeylanica (1.62% w/w) followed by the roots of P. indica (0.97% w/w) and then P. auriculata (0.33-0.53% w/w). In contrast, plumbagin was not detected in the stems and leaves of P. indica and in the leaves of P. auriculata, whereas very low concentrations (<0.02% w/w) of plumbagin were detected in the stems and leaves of P. zeylanica and in the stems of P. auriculata. HPTLC fingerprints of the leaf and root of the three species exhibited distinguishable profiles, while those of the stems were undifferentiated.

Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Microscopy; Naphthoquinones; Phytotherapy; Plant Leaves; Plant Roots; Plant Stems; Plants, Medicinal; Plumbaginaceae; Spectrophotometry, Ultraviolet

The peroxide stress response regulator PerR coordinates the oxidative-stress defenses of group A Streptococcus (GAS). We now show that PerR is expressed from an operon encoding a putative DNA polymerase I (PolA1), among other GAS products. A polA1 deletion mutant exhibited wild-type growth but showed reduced capacity to repair DNA damage caused by UV light or ciprofloxacin. Mutant bacteria were hypersensitive to H(2)O(2), compared with the wild type or a complemented mutant strain, and remained severely attenuated even after adaptation at sublethal H(2)O(2) levels, whereas wild-type bacteria could adapt to withstand peroxide challenge under identical conditions. The hypersensitivity of the mutant was reversed when bacteria were grown in iron-depleted medium and challenged in the presence of a hydroxyl radical scavenger, results that indicated sensitivity to hydroxyl radicals generated by Fenton chemistry. The peroxide resistance of a perR polA1 double mutant following adaptation at sublethal H(2)O(2) levels was decreased 9-fold relative to a perR single mutant, thus implicating PolA1 in PerR-mediated defenses against peroxide stress. Cultures of the polA1 mutant grown with or without prior H(2)O(2) exposure yielded considerably lower numbers of rifampin-resistant mutants than cultures of the wild type or the complemented mutant strain, a finding consistent with PolA1 lacking proofreading activity. We conclude that PolA1 promotes genome sequence diversity while playing an essential role in oxidative DNA damage repair mechanisms of GAS, dual functions predicted to confer optimal adaptive capacity and fitness in the host. Together, our studies reveal a unique genetic and functional relationship between PerR and PolA1 in streptococci.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Ciprofloxacin; DNA Damage; DNA Polymerase I; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Genetic Variation; Genome, Bacterial; Hydrogen Peroxide; Mutation; Naphthoquinones; Repressor Proteins; Rifampin; Streptococcus pyogenes; Ultraviolet Rays

Paramphistomiasis causes enteritis and anemia in livestocks and result in substantial production and economic losses. It is considered a neglected tropical disease, with no effective trematodicidal compound for treatment. Plumbagin (PB), a compound founds to be rich in the roots of Plumbago indica, is a naphthoquinone derivatives which can induce oxidative stress in parasites. In this study we have evaluated the anthelmintic activity of PB against adult Paramphistomum cervi by incubating the parasites in M-199 medium containing 0.1, 1.0, 10 and 100 μg/ml of the PB, and albendazole (ABZ) at the concentration of 100 μg/ml as the positive control, for 3, 6, 12 and 24 h, using relative motility (RM) assay and observed by scanning electron microscopy (SEM). After 12 h exposure with 100 μg/ml ABZ, flukes showed decreased contraction and motility. At 24 h incubation they showed only active movement of some part of the body. The PB-treated flukes at all concentrations showed rapid decrease of motility at 3 h incubation. In 0.1, 1.0 and 10 μg/ml of PB, the RM values were decreased sharply from 3 to 12 h, and then they were killed since 12 h in the incubation with 10 μg/ml of PB. The highest parasite mortality was found as early as 3h when they were incubated with 100 μg/ml of PB. The morphological changes on the tegumental surface were similar in both flukes treated with ABZ and PB, which sequentially comprised of swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesions were formed which exposed the basal lamina. The damage appeared more severe on the ventral than the dorsal surface, and earlier on the anterior part and lateral margins of middle third when compared to the posterior part of the parasites's bodies. The severity and rapidity of the damages were enhanced with increasing concentration of PB, which showed stronger activity than ABZ. Hence, PB has a potential to be an anthelmintic drug against adult P.cervi.

Topics: Albendazole; Animals; Anthelmintics; Cattle; Microscopy, Electron, Scanning; Movement; Naphthoquinones; Paramphistomatidae; Rumen

Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated.. Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma-optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes.. The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes.. The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Arthropods; Chromatography, Ion Exchange; Escherichia coli; Genes, rRNA; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Naphthoquinones; Nitrogen; Plant Exudates; Plant Proteins; Protein Structure, Secondary; Pseudomonas syringae; RNA, Ribosomal, 16S; Saccharomyces cerevisiae; Sarraceniaceae; Species Specificity

We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2 × 10(6)) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly (p = 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver (p = 0.037), but inhibition of metastasis into the lungs (p = 0.60) and lymph nodes (p = 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes (p = 0.034) and lungs (p = 0.028), and a trend to significance in liver (p = 0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKCε, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and Bcl(xL)), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Naphthoquinones; Neoplasm Metastasis; Nitric Oxide Synthase Type II; Plumbaginaceae; Prostate; Prostatic Neoplasms; Protein Kinase C; STAT3 Transcription Factor

Diospyros bipindensis (Gürke) stem bark is used in Cameroon by Baka Pygmies for the treatment of respiratory disorders.. To assess the anti-inflammatory, antibacterial and antioxidant properties of constituents from the bark extracts through bioassay-guided fractionation.. The anti-inflammatory activity of extracts, fractions and pure compounds was assessed through the inhibition of the pro-inflammatory mediator nuclear factor-kappa B (NF-κB) transcriptional activity and nitric oxide (NO) production. DPPH, ABTS and ORAC assays were used for determining the antioxidant properties. The activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Klebsiella pneumoniae, was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the macrodilution method.. The water extract showed antimicrobial activity against S. pneumoniae (MIC: 300 μg/ml) and S. pyogenes (MIC: 300 μg/ml). The dichloromethane extract efficiently inhibited NF-κB transcriptional activity and NO production and exhibited significant antioxidant activity in the ORAC assay. An interesting activity was also found against S. pneumoniae (MIC: 200 μg/ml), S. aureus (MIC: 400 μg/ml) and S. pyogenes (MIC: 200 μg/ml). The phytochemical investigation of the dichloromethane extract afforded plumbagin, canaliculatin, ismailin, betulinic acid and 4-hydroxy-5-methyl-coumarin as the main constituents. Plumbagin and ismailin were found to be responsible for the main biological activities observed.. These results may provide a rational support for the traditional use of Diospyros bipindensis stem bark in the treatment of respiratory disorders, since the anti-inflammatory, antimicrobial and antioxidant compounds isolated from the dichloromethane extract were also present in the traditional water extract.

Topics: Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Antioxidants; Bacteria; Betulinic Acid; Biphenyl Compounds; Cell Line; Diospyros; HEK293 Cells; Humans; Mice; Microbial Sensitivity Tests; Naphthoquinones; NF-kappa B; Nitric Oxide; Pentacyclic Triterpenes; Picrates; Plant Bark; Plant Extracts; Triterpenes; Tumor Necrosis Factor-alpha

Benjakul [BEN], a Thai Traditional medicine preparation, is composed of five plants: Piper chaba fruit [PC], Piper sarmentosum root [PS], Piper interruptum stem [PI], Plumbago indica root [PL] and Zingiber officinale rhizome [ZO]. From selective interviews of folk doctors in Southern Thailand, it was found that Benjakul has been used for cancer patients.. To investigate cytotoxicity activity of Benjakul preparation [BEN] and its ingredients against three human cancer cell lines, large lung carcinoma cell line (COR-L23), cervical cancer cell line (Hela) liver cancer cell line (HepG2) as compared with normal lungfibroblast cell (MRC-5) by using SRB assay.. The extraction as imitated the method used by folk doctors was done by maceration in ethanol and boiling in water Bioassay guided isolation was used isolated cytotoxic compound.. The ethanolic extracts of PL, ZO, PC, PS, BEN and PS showed specific activity against lung cancer cell (IC50 = 3.4, 7.9, 15.8, 18.4, 19.8 and 32.91 microg/ml) but all the water extracts had no cytotoxic activity. Three active ingredients [6-gingerol, plumbagin and piperine as 0.54, 4.18 and 7.48% w/w yield of crude extract respectively] were isolated from the ethanolic extract of BEN and they also showed cytotoxic activity with plumbagin showing the highest cytotoxic activity against COR-L23, HepG2, Hela and MRC-5 (IC50 = 2.55, 2.61, 4.16 and 11.54 microM respectively).. These data results may support the Thai traditional doctors who are using Benjakul to treat cancer patients and three of its constituents (6-gingerol, plumbagin and piperine) are suggested to be used as biomarkers for standardization of this preparation.

Topics: Alkaloids; Benzodioxoles; Catechols; Cell Line, Tumor; Fatty Alcohols; Female; Humans; Liver Neoplasms; Lung Neoplasms; Medicine, East Asian Traditional; Naphthoquinones; Phytotherapy; Piper; Piperidines; Plant Extracts; Plants, Medicinal; Plumbaginaceae; Polyunsaturated Alkamides; Thailand; Uterine Cervical Neoplasms; Zingiber officinale

Angiogenesis-based therapy is an effective anti-tumour strategy and previous reports have shown some beneficial effects of a naturally occurring bioactive compound plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone). Here, we sought to determine the biological effects of plumbagin on signalling mechanisms during tumour angiogenesis.. The effects of plumbagin were evaluated in various in vitro assays which utilised human umbilical vein endothelial cells (HUVEC) proliferation, migration and tube formation. Plumbagin was also evaluated in vivo using chicken embryo chorioallantoic membrane (CAM) and mouse corneal micropocket models., Human colon carcinoma and prostate cancer xenograft mouse models were used to evaluate the effects of plumbagin on angiogenesis. Immunofluorescence, GST pull-down and Western blotting were employed to explore the underlying mechanisms of VEGF receptor (VEGFR)2-mediated Ras signalling pathways.. Plumbagin not only inhibited endothelial cell proliferation, migration and tube formation but also suppressed chicken chorioallantoic membrane neovascularzation and VEGF-induced mouse corneal angiogenesis. Moreover, plumbagin suppressed tumour angiogenesis and tumour growth in human colon carcinoma and prostate cancer xenograft mouse models. At a molecular level, plumbagin blocked the Ras/Rac/cofilin and Ras/MEK signalling pathways mediated by VEGFR2 in HUVECs.. Plumbagin inhibited tumour angiogenesis and tumour growth by interference with the VEGFR2-mediated Ras signalling pathway in endothelial cells. Our findings demonstrate a molecular basis for the effects of plumbagin and suggest that this compound might have therapeutic ant-tumour effects.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chickens; Chorioallantoic Membrane; Human Umbilical Vein Endothelial Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Nude; Naphthoquinones; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; ras Proteins; Signal Transduction; Tumor Burden; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Trypanothione and trypanothione reductase (TryR)-based redox metabolism found in Leishmania and other trypanosomatids exemplify the unique features of this group of organisms. Its absence in mammalian hosts, together with the sensitivity of trypanosomes against oxidative stress, makes this enzyme a unique target for exploitation for potential antileishmanial chemotherapeutics. Plumbagin, a plant-derived naphthoquinone, is reported to possess antileishmanial properties by inhibiting TryR. We here report the kinetics of the inhibitory mechanism of plumbagin and its derivative, 2-methoxy 1, 4-naphthoquinone. Interestingly, apart from acting as inhibitor, these compounds also act as subversive substrates and subvert the physiological function of enzyme by converting it from an antioxidant to a prooxidant. Both naphthoquinones show a significant effect on redox homeostasis and results in increased reactive oxygen species, resulting in morphological changes and parasite death.

Topics: Antiprotozoal Agents; Cell Survival; Enzyme Inhibitors; Leishmania; NADH, NADPH Oxidoreductases; Naphthoquinones; Oxidation-Reduction; Sulfhydryl Compounds

A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.

Topics: Agrobacterium; Naphthoquinones; Plant Roots; Plants, Genetically Modified; Plumbaginaceae; Recombinant Proteins; Single-Chain Antibodies

Osteosarcoma, which is the most common primary bone tumor, occurs most frequently in adolescents. A number of studies have indicated that plumbagin (PL) (5-hydroxy-2-methyl-1, 4-naphthoquinone), a compound found in the plants of the Plumbaginaceae and Droseraceae families, possesses anticancer activity. However, its anticancer effects and mechanisms against osteosarcoma have not been explored. To determine the anticancer effect of PL on osteosarcoma cell lines MG-63 and U2OS, cell viability, apoptosis, cell cycle distribution, caspase-3 and caspase-9 activity and intracellular reactive oxygen species (ROS) generation were measured, and Western blot analyses were performed. PL significantly inhibited the growth of osteosarcoma cells, particularly U2OS cells. PL up-regulated the expression of p53 in U2OS cells and p21 in the two osteosarcoma cell lines causing cell cycle arrest by decreasing the expression of murine double minute 2 (MDM2)/cyclin B1 and cyclin D1. Furthermore, PL altered the ratio of Bax/Bcl-2, and may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and caspase-9 activation. We also found that PL induced the generation of ROS in osteosarcoma cell lines. To conclude, PL exerted anticancer activity on osteosarcoma cells by inducing pro-apoptotic signaling and modulating the intracellular ROS that causes induction of apoptosis. These effects may relate to the p53 status.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Humans; Naphthoquinones; Osteosarcoma; Proto-Oncogene Proteins c-mdm2; Reactive Oxygen Species; Tumor Suppressor Protein p53

Three naphthoquinones, plumbagin (1), 3,3'-biplumbagin (2) and elliptinone (3), isolated from Plumbago indica roots by antibacterial bioassay-guided isolation, were used as standard markers for quantitative determination. A reversed-phase HPLC method was established for the simultaneous determination of the naphthoquinones in P. indica root extracts. The method utilised a Phenomenex® ODS column (4.6 × 150 mm, 5 µm) at 25°C with a mixture of methanol and 5% aqueous acetic acid (80 : 20 v/v) as the mobile phase at a flow rate of 0.85 mL/min, and UV detection at 260 nm. The parameters of linearity, precision, accuracy specificity and sensitivity of the method were evaluated. The recovery of the method was 98.6-100.6% with good linearity (r (2 )≥ 0.9997) for all three naphthoquinones. A high degree of sensitivity, specificity as well as repeatability and reproducibility (R.S.D. values less than 5%) were also achieved.

Topics: Anti-Bacterial Agents; Chromatography, High Pressure Liquid; Limit of Detection; Naphthoquinones; Plant Extracts; Plant Roots; Plumbaginaceae; Propionibacterium acnes; Sensitivity and Specificity; Staphylococcus aureus

Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone, was isolated from the roots of the Plumbago zeylanica L. (also known as Chitrak). The roots of P. zeylanica L. have been used in Indian medicine for >2500 years as an anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective agent. We present here that topical application of non-toxic doses (100-500 nmol) of PL to skin elicits dose-dependent inhibition of ultraviolet radiation (UVR)-induced development of squamous cell carcinomas (SCC). In this experiment, FVB/N mice were exposed to UVR (2 kJ/m(2)) three times weekly from a bank of six Kodacel-filtered FS40 sunlamps (∼ 60% UVB and 40% UVA). Carcinoma incidence in mice treated with vehicle, 100, 200 or 500 nmol PL, at 44 weeks post-UVR, were 86, 80 (P = 0.67), 53 (P = 0.12) and 7% (P = 0.0075), respectively. Both vehicle and PL-treated mice gained weight and did not exhibit any signs of toxicity during the entire period of the experiment. Molecular mechanisms associated with inhibition of UVR-induced development of SCC involved induction of apoptosis and inhibition of cell proliferation. Specific findings are that PL treatment (i) inhibited UVR-induced DNA binding of activating protein-1, nuclear factor-kappaB, Stat3 transcription factors and Stat3-regulated molecules (cdc25A and Survivin); (ii) inhibited protein levels of pERK1/2, PI3K85, pAKTSer473, Bcl(2), BclxL, proliferating cell nuclear antigen and cell cycle inhibitory proteins p27 and p21 and (iii) increased UVR-induced Fas-associated death domain expression, poly (ADP-ribose) polymerase protein cleavage and Bax/Bcl(2) ratio. Taken together, our findings suggest that PL may be a novel agent for the prevention of skin cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Squamous Cell; Cell Survival; DNA; Female; Mice; Naphthoquinones; Neoplasms, Radiation-Induced; NF-kappa B; Skin Neoplasms; STAT3 Transcription Factor; Transcription Factor AP-1; Ultraviolet Rays

Bone loss is one of the major complications of advanced cancers such as breast cancer, prostate cancer, and multiple myeloma; agents that can suppress this bone loss have therapeutic potential. Extensive research within the last decade has revealed that RANKL, a member of the tumor necrosis factor superfamily, plays a major role in cancer-associated bone resorption and thus is a therapeutic target. We investigated the potential of vitamin K3 analogue plumbagin (derived from Chitrak, an Ayurvedic medicinal plant) to modulate RANKL signaling, osteoclastogenesis, and breast cancer-induced osteolysis. Plumbagin suppressed RANKL-induced NF-κB activation in mouse monocytes, an osteoclast precursor cell, through sequential inhibition of activation of IκBα kinase, IκBα phosphorylation, and IκBα degradation. Plumbagin also suppressed differentiation of these cells into osteoclasts induced either by RANKL or by human breast cancer or human multiple myeloma cells. When examined for its ability to prevent human breast cancer-induced bone loss in animals, plumbagin (2 mg/kg body weight) administered via the intraperitoneal route significantly decreased osteolytic lesions, resulting in preservation of bone volume in nude mice bearing human breast tumors. Overall, our results indicate that plumbagin, a vitamin K analogue, is a potent inhibitor of osteoclastogenesis induced by tumor cells and of breast cancer-induced osteolytic metastasis through suppression of RANKL signaling.

Topics: Animals; Antineoplastic Agents, Phytogenic; Blotting, Western; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line; Cell Line, Tumor; Female; Humans; I-kappa B Proteins; Macrophages; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; NF-kappa B; NF-KappaB Inhibitor alpha; Osteoclasts; Osteogenesis; Osteolysis; RANK Ligand; Signal Transduction; Xenograft Model Antitumor Assays

To evaluate the biological effects of static magnetic fields (SMFs) up to 13 Tesla (T), with respect to superoxide behavior, by determining the effect on mutagenicity in superoxide dismutase (SOD)-deficient Escherichia coli strain QC774, and its parental strain GC4468.. Experimental strains were exposed to a 5, 10, or 13T SMF for 24 h at 37°C in Luria-Bertani medium. To evaluate mutagenicity after SMF exposure, the mutation frequency in thymine synthesis genes was determined. The effect of exposure to a 5 or 13T SMF on mutagenicity induced by plumbagin was also investigated.. No statistically significant differences in the mutation frequency in thymine synthesis genes were observed between SMF-exposed cells and unexposed cells at any of the applied magnetic flux densities. Furthermore, exposure to SMFs up to 13T did not affect mutagenicity induced by plumbagin.. Exposure to SMFs up to 13T caused neither mutagenicity nor co-mutagenicity in the SOD-deficient E. coli strain QC774 or in its parental strain GC4468, suggesting that exposure to strong SMFs does not affect the behavior of superoxides in these microorganisms.

Topics: Cell Survival; Escherichia coli; Magnetic Fields; Mutagenesis; Naphthoquinones; Superoxide Dismutase; Thymine

The purpose of this study is to elucidate the possible mechanism of superoxide formation through redox cycling of plumbagin (PLG) in pig heart. Of four 1,4-naphthoquinones tested in this study, PLG was most efficiently reduced in the cytosolic fraction of pig heart. On the other hand, lawsone (LAS) was little reduced. Thus, whether or not PLG and LAS induce the formation of superoxide anion radical in pig heart cytosol was examined, by using the methods of cytochrome c reduction and chemiluminescence. PLG significantly induced the formation of superoxide anion radical, even though LAS had no ability to mediate superoxide formation. PLG was a significant inhibitor for the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by tetrameric carbonyl reductase (TCBR) in pig heart cytosol. Furthermore, PLG was confirmed to competitively inhibit the 4-BP reduction, and the optimal pH for the PLG reduction was around 6.0 similar to that for the 4-BP reduction. These results suggest that PLG mediates superoxide formation through its redox cycling involved in the two-electron reduction catalyzed by TCBR, and induces oxidative stress in pig heart.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cytochromes c; Myocardium; Naphthoquinones; Oxidation-Reduction; Pyridines; Superoxides; Swine

To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells.. Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-κB-regulated gene products and TNF-α-induced activation of p65, IκBα, and IKK. The intracellular location of NF-κB p65 was detected using confocal microscopy.. Plumbagin (2.5-40 μmol/L) concentration-dependently reduced the viability of the GC cells. The IC(50) value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 μmol/L, respectively. The compound (5-20 μmol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-αand cisplatin. The compound (10 μmol/L) downregulated the expression of NF-κB-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-κB p65 nuclear translocation, the compound also suppressed TNF-α-induced phosphorylation of p65 and IKK, and the degradation of IκBα.. Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-κB pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; I-kappa B Kinase; Naphthoquinones; NF-kappa B; Plumbaginaceae; Signal Transduction; Stomach Neoplasms; Tumor Necrosis Factor-alpha

Barleria alluaudii and Diospyros maritima were both investigated as part of an ongoing search for synergistic TRAIL (tumor necrosis factor-α-related apoptosis-inducing ligand) sensitizers. As a result of this study, two naphthoquinone epoxides, 2,3-epoxy-2,3-dihydrolapachol (1) and 2,3-epoxy-2,3-dihydro-8-hydroxylapachol (2), both not previously isolated from natural sources, and the known 2-methylanthraquinone (3) were identified from B. alluaudii. Time-dependent density functional theory (TD-DFT) calculations of electronic circular dichroism (ECD) spectra were utilized to establish the absolute configuration of 1 and 2. Additionally, five known naphthoquinone derivatives, maritinone (4), elliptinone (5), plumbagin (6), (+)-cis-isoshinanolone (7), and ethylidene-6,6'-biplumbagin (8), were isolated from D. maritima. Compounds 1, 2, and 4-6 showed varying levels of synergy with TRAIL. Maritinone (4) and elliptinone (5) showed the highest synergistic effect, with more than a 3-fold increase in activity observed with TRAIL than with compound alone.

Topics: Acanthaceae; Anthraquinones; Diospyros; Madagascar; Molecular Structure; Naphthoquinones; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Pancreatic cancer (PC) is the most aggressive malignant disease, ranks as the fourth most leading cause of cancer-related death among men and women in the United States. We present here that plumbagin (PL), a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L, inhibits the growth of PC cells both in vitro and in vivo model systems. PL treatment induces apoptosis and inhibits cell viability of PC cells (PANC1, BxPC3 and ASPC1). In addition, i.p. administration of PL (2 mg/kg body weight, 5 days a week) in severe combined immunodeficiency (SCID) mice beginning 3 days after ectopic implantation of PANC1 cells resulted in a significant (P < 0.01) inhibition of both tumor weight and volume. PL treatment inhibited (1) constitutive expression of epidermal growth factor receptor (EGFR), pStat3Tyr705 and pStat3Ser727, (2) DNA binding of Stat3 and (3) physical interaction of EGFR with Stat3, in both cultured PANC1 cells and their xenograft tumors. PL treatment also inhibited phosphorylation and DNA-binding activity of NF-κB in both cultured PC cells (PANC1 and ASPC1) and in PANC1 cells xenograft tumors. Downstream target genes (cyclin D1, MMP9 and Survivin) of Stat3 and NF-κB were similarly inhibited. These results suggest that PL may be used as a novel therapeutic agent against human PC. Published 2012 Wiley-Liss, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; ErbB Receptors; Humans; Inhibitor of Apoptosis Proteins; Male; Matrix Metalloproteinase Inhibitors; Mice; Mice, SCID; Naphthoquinones; NF-kappa B; Pancreatic Neoplasms; Phosphorylation; Plant Extracts; Signal Transduction; STAT3 Transcription Factor; Survivin

Novel plumbagin hydrazonates were prepared, structurally characterized and evaluated for anti-proliferative activity against estrogen receptor-positive MCF-7 and triple negative MDA-MB-231 and MDA-MB-468 breast cancer cell lines which exhibited superior inhibitory activity than parent plumbagin compound. Molecular docking studies indicated that hydroxyl groups on plumbagin and hydrazonate side chain favor additional hydrogen bonding interactions with amino acid residues in p50-subunit of NF-κB protein and these compounds inhibited NF-κB expression which may be responsible for the enhanced anti-proliferative activity. These compounds were found to be more effective against triple negative breast cancer cells and might serve as a starting point for building future strategies against triple negative breast cancers which are known for their increased drug resistance and poor prognosis of breast cancer patients.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Hydrazones; Hydrogen Bonding; Naphthoquinones; NF-kappa B; Protein Binding; Structure-Activity Relationship

Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein Receptors, Type II; Calcium; Cardiotonic Agents; Cell Proliferation; Cells, Cultured; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; In Situ Nick-End Labeling; Interleukin-6; Male; Muscle, Smooth, Vascular; Naphthoquinones; NFATC Transcription Factors; Potassium Channels, Voltage-Gated; Proliferating Cell Nuclear Antigen; Rats; rho-Associated Kinases

Breast cancer is the leading cause of death-related cancers in women. Approximately 30% of breast cancers overexpress the Her2 oncogene, which is associated with a poor prognosis and increased resistance to chemotherapy. Plumbagin (1), a constituent of species in the plant genera Drosera and Plumbago, displays antineoplastic activity toward various cancers. The present study was aimed at determining the anticancer potential of 1 toward Her2-overexpressing breast cancer cells and defining the mode of cell death induced in these cells. The results showed that 1 exhibited high antiproliferative activity toward the Her2-overexpressing cell lines SKBR3 and BT474. The antiproliferative activity of 1 was associated with apoptosis-mediated cell death, as revealed by caspase activation and an increase in the sub-G1 fraction of the cell cycle. Compound 1 increased the levels of the proapoptotic Bcl-2 family of proteins and decreased the level of the antiapoptotic Bcl-2 protein in SKBR3 and BT474 cells. Thus, these findings indicate that 1 induces apoptosis in Her2-overexpressing breast cancers through the mitochondrial-mediated pathway and suggest its potential for further investigation for the treatment of Her2-overexpressing breast cancer.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Cycle; Female; Humans; Mitochondria; Naphthoquinones; Receptor, ErbB-2

The aim of this study was to investigate the growth-inhibiting efficacy of Drosera intermedia extracts (water, methanol and n-hexane) against four food spoilage yeasts and five filamentous fungi strains responsible for food deterioration and associated with mycotoxin production, in order to identify potential antimycotic agents.. The n-hexane extract showed a broad activity spectrum against all tested microorganisms, followed, in activity, by the methanol and water extracts. The major component of the n-hexane extract was purified using a solid-phase extraction column and identified as plumbagin. Results show that high-purity plumbagin can be produced from D. intermedia cultures following a simple and effective isolation procedure. A sample of purified plumbagin was tested against the same panel of microorganisms and high growth-inhibiting capacity was observed. Minimum inhibitory concentrations less than 2 µg mL(-1) were obtained against the filamentous fungi. In the case of the species Aspergillus fumigatus, A. niger and A. flavus, activities comparable to miconazole were obtained.. The results obtained provided evidence of the antimycotic activity of plumbagin, suggesting that D. intermedia could be the source of an interesting compound for the food industry as an alternative to preservatives.

Topics: Antifungal Agents; Aspergillus; Drosera; Food Microbiology; Food Preservation; Fungi; Humans; Miconazole; Microbial Sensitivity Tests; Naphthoquinones; Plant Extracts; Yeasts

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-κB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-κB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.

Topics: Actins; Animals; Antigens, Differentiation; Antineoplastic Agents; Bone and Bones; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cytokines; Enzyme Activation; Female; Gene Expression; Humans; Inhibitory Concentration 50; Macrophages; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Naphthoquinones; Neoplasm Invasiveness; NF-kappa B; Osteoclasts; Osteolysis; Protein Binding; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; TNF Receptor-Associated Factor 6; Tumor Microenvironment; Xenograft Model Antitumor Assays

Various shortcomings of the available methods of extraction of plumbagin from Plumbago zeylanica using non-edible organic solvents coupled with the poor aqueous solubility and low bioavailability called for extracting plumbagin in a water soluble form via a single step technique using hydrophilic lipid Gelucire 44/14.. Gelucire extract of P. zeylanica (GPZ) was prepared and evaluated for extraction efficiency, High-performance thin layer chromatography (HPTLC) and thermal analysis. In vitro intestinal absorption and bioavailability of plumbagin from GPZ in comparison with that of aqueous (APZ), ethanolic extract (EPZ) and standard plumbagin studied using non-everted rat intestinal sac model.. The GPZ showed significantly higher extraction efficiency (3.24±0.12% w/w) compared to ethanolic (EPZ) and aqueous (APZ) extraction, 2.48±0.16% w/w and 0.07±0.02% w/w respectively. GPZ displayed significantly higher Q(30min) (cumulative percentage absorption of plumbagin in 30 min) and lower t(40%) (time required for 40% w/w drug absorption). The flux and apparent permeability coefficient in duodenum and ileum were 2, 3 and 6 fold higher than EPZ, standard plumbagin and APZ respectively.. Improved therapeutic efficacy of plumbagin may be due to the micellar solubilization and consequent enhanced partitioning of plumbagin through intestinal by Gelucire which was reflected in the in vivo anti-inflammatory study conducted in rats.. Thus extraction using Gelucire can be proclaimed as an efficient, economic and solvent-free technique for extraction of plumbagin and can be utilized for various clinically important water insoluble phytoconstituents in order to improve their biopharmaceutical properties.

Topics: Animals; Carrageenan; Chemical Fractionation; Chromatography, High Pressure Liquid; Inflammation; Intestinal Absorption; Intestinal Mucosa; Intestines; Liquid-Liquid Extraction; Male; Mice; Models, Biological; Naphthoquinones; Permeability; Plant Extracts; Plumbaginaceae; Polyethylene Glycols; Rats; Toxicity Tests, Acute; Water

While the 1,4-naphthoquinone derivatives 7-methyljuglone (1) and plumbagin (2) possess a diverse and well documented array of biological activities, relatively little remains known about the functional significance of these compounds in planta and, in particular, their possible relation to carnivorous syndromes. In addition, the chemotaxonomic distribution of naphthoquinones (NQs) amongst species of Drosera L. is of phytopharmaceutical interest. Following the quantitative assessment of interspecific variation of 1 and 2 in 13 species and cultivars of Drosera, our findings demonstrate that these NQs are ubiquitously coproduced in, generally, species-specific ratios, and that 1 appears negatively associated with the occurrence of pigmentation in sundews. The prospective antifeedant function of 1 was evaluated in relation to allocation in various organs and ontogenetic phases of D. capensis L., revealing that significantly higher levels were accumulated in young and reproductive organs, most likely for defensive purposes. Investigation into the relationship between the biosynthesis of NQs and carnivory showed that production of 1 is optimally induced and localized in leaves in response to capture of insect prey. As a whole, these findings reveal the clear importance of this secondary metabolite in ecological interactions as well as holding implication for future bioactivity studies on the genus.

Topics: Drosera; Ecological and Environmental Phenomena; Naphthoquinones; Plant Extracts

Chemical investigatation of Drosera peltata var. multisepala led to the isolation of eleven compounds using various chromatographic techniques. The structures of these compounds were elucidated as isoshinanolone-4-O-beta-D-glucoside (1), isoshinanolone (2), epi-isoshinanolone (3), plumbagin (4), droserone (5), droserone-5-O-glucoside (6), quercetin (7), kaempferol (8) , gossypetin-8-O-glucoside (9), 3,3'-dimethoxy ellagic acid (10), and ellagic acid (11) by their physicochemical properties and spectral data analysis. Compound 1 was a new compound. Compounds 3, 8, 10, and 11 were isolated from this plant for the first time.

Topics: Chromatography, Liquid; Drosera; Drugs, Chinese Herbal; Ellagic Acid; Glucosides; Kaempferols; Magnetic Resonance Spectroscopy; Molecular Structure; Naphthoquinones; Plant Extracts; Quercetin; Spectrometry, Mass, Electrospray Ionization; Tetrahydronaphthalenes

Histone modifications; acetylation, methylation (both Lysine and Arginine) etc., at different positions regulates the chromatin fluidity and function in a combinatorial manner, which could be referred as an epigenetic language. In the context of transcription, histone acetylation, methylation and phosphorylation at specific sites, especially at the N-terminal tails of histones play very important roles in activation and/or repression. While acetylation of histones is generally important for transcriptional activation, methylation and phosphorylation could also be involved in repression, depending on the context. Here, we have investigated the crosstalk of histone modifications on a gross scale over histone H3, using a small molecule inhibitor of lysine acetyltransferase KAT3B/p300, Plumbagin, to analyze the histone modification profile upon inhibition of acetylation. In addition to the inhibition of acetylation, there was a concomitant decrease of transcriptional activation mark, H3 lysine 4 trimethylation (H3K4me3) in the cellular context. The histone H3 Serine 10 Phosphorylation (H3S10p) also decreased upon inhibition of acetylation. However, there were no changes observed with transcriptional repressive marks like H3 Lysine 9 di/trimethylation (H3K9me2/me3) suggesting that transcriptional activation marks were selectively targeted. These data suggest that Plumbagin induces a distinct modification profile involving transcriptional activation marks H3K4me3 and H3S10 phosphorylation in the context of histone acetylation brought about by KAT3B/ p300.

Topics: Acetylation; Acetyltransferases; Apoptosis; Cell Survival; Cells, Cultured; Enzyme Inhibitors; HEK293 Cells; Histones; Humans; Methylation; Molecular Structure; Molecular Weight; Naphthoquinones; Phosphorylation; Structure-Activity Relationship

Plumbago zeylanica L. root is widely used in Indian medicine to treat diabetes mellitus. The aim of the present investigation was to evaluate the antidiabetic effects of plumbagin isolated from P. zeylanica L. root and its effect on GLUT4 translocation in STZ-induced diabetic rats. Plumbagin (15 and 30 mg/kg b wt) was orally administered to STZ-induced diabetic rats for 28 days. An oral glucose tolerance test was performed on 21st day. The effect of plumbagin on body weight, blood glucose, plasma insulin, total protein, urea, creatinine, liver glycogen, plasma enzymes (SGOT, SGPT and ALP) and carbohydrate metabolism enzymes (glucose-6-phosphatase, fructose-1,6-bisphosphatase and hexokinase) were investigated. GLUT4 mRNA and protein expression in skeletal muscles were also studied. Plumbagin significantly reduced the blood glucose and significantly altered all other biochemical parameters to near normal. Further, plumbagin increased the activity of hexokinase and decreased the activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase significantly in treated diabetic rats. Enhanced GLUT4 mRNA and protein expression were observed in diabetic rats after treatment with plumbagin. The results indicated that plumbagin enhanced GLUT4 translocation and contributed to glucose homeostasis. It could be further probed for use as a drug to treat diabetes.

Topics: Animals; Blood Glucose; Blotting, Western; Body Weight; Creatinine; Diabetes Mellitus, Experimental; Fructose-Bisphosphatase; Gene Expression Regulation; Glucose Tolerance Test; Glucose Transporter Type 4; Glucose-6-Phosphatase; Hexokinase; Hypoglycemic Agents; Insulin; Liver Glycogen; Male; Muscle, Skeletal; Naphthoquinones; Plant Extracts; Plant Roots; Plumbaginaceae; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Toxicity Tests, Acute

Plumbagin (PL), 5-hydroxy-2-methyl-1,4-naphthoquinone, is a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L. (also known as chitrak). PL has also been found in Juglans regia (English Walnut), Juglans cinerea (whitenut) and Juglans nigra (blacknut). The roots of P. zeylanica have been used in Indian and Chinese systems of medicine for more than 2500 years for the treatment of various types of ailments. We were the first to report that PL inhibits the growth and invasion of hormone refractory prostate cancer (PCa) cells [Aziz,M.H. et al. (2008) Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res., 68, 9024-9032.]. Now, we present that PL inhibits in vivo PCa development in the transgenic adenocarcinoma of mouse prostate (TRAMP). PL treatment (2 mg/kg body weight i.p. in 0.2 ml phosphate-buffered saline, 5 days a week) to FVB-TRAMP resulted in a significant (P < 0.01) decrease in prostate tumor size and urogenital apparatus weights at 13 and 20 weeks. Histopathological analysis revealed that PL treatment inhibited progression of prostatic intraepithelial neoplasia (PIN) to poorly differentiated carcinoma (PDC). No animal exhibited diffuse tumor formation in PL-treated group at 13 weeks, whereas 75% of the vehicle-treated mice elicited diffuse PIN and large PDC at this stage. At 20 weeks, 25% of the PL-treated animals demonstrated diffuse PIN and 75% developed small PDC, whereas 100% of the vehicle-treated mice showed large PDC. PL treatment inhibited expression of protein kinase C epsilon (PKCε), signal transducers and activators of transcription 3 phosphorylation, proliferating cell nuclear antigen and neuroendocrine markers (synaptophysin and chromogranin-A) in excised prostate tumor tissues. Taken together, these results further suggest PL could be a novel chemopreventive agent against PCa.

Topics: Adenocarcinoma; Animals; Anticarcinogenic Agents; Antigens, Polyomavirus Transforming; Chromogranin A; Disease Models, Animal; Male; Mice; Mice, Transgenic; Naphthoquinones; Phosphorylation; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Kinase C-epsilon; STAT3 Transcription Factor; Synaptophysin

Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI(50); 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Death; Cell Proliferation; Cell Survival; Down-Regulation; Drug Screening Assays, Antitumor; Female; Gene Knockdown Techniques; Haploinsufficiency; Heterozygote; Humans; MCF-7 Cells; Models, Biological; Mutation; Naphthoquinones; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; RNA, Small Interfering; Schizosaccharomyces

Invasive lobular carcinoma (ILC) and its variants represent 5% to 15% of all invasive breast cancers diagnoses annually. AS a serine/threonine kinase, mammalian target of rapamycin (mTOR) is often a downstream effector of PI3K/Akt (phosphatidyl inositol 3-kinase/protein kinase B) signaling pathway in breasts and many types of cancer cells. Therefore, agents that target mTOR in direct or indirect manner are being developed in anti-cancer therapy.. In this study, our objective here was to explore more crosstalk pathway with mTOR signaling pathway.. We collected pathways data from published database, then based on bioinformatics methods we analyzed the significant pathways in the database, additionally, the crosstalk pathways were also analyzed which were defined as those pathways which have the overlapping genes with each other.. As we expected, the results showed that Notch signaling pathway (hsa04330), Regulation of autophagy (hsa04140), and Adipocytokine signaling pathway (hsa04920) were linked to mTOR signaling pathway. All of them have been demonstrated participate in breast cancer progression.. We obtained some key pathways that crosstalked with mTOR signaling pathway, we hope our study could provide novel therapeutic approaches for breast cancer.

Topics: Adipokines; Autophagy; Breast Neoplasms; Carcinoma, Lobular; Female; Humans; Naphthoquinones; Signal Transduction; TOR Serine-Threonine Kinases

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, 1), a natural product from plants with potential anticancer potency, induces apoptosis. Mechanisms involved in 1-induced apoptosis include mitochondrial depolarization, inactivation of NF-κB, and altered expression of anti- and proapoptotic Bcl proteins. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which, like apoptosis, results in cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)]i) and ceramide formation. The present study explored whether 1 stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo-3 fluorescence, and ceramide abundance utilizing antibodies. A 48 h exposure to 1 (2 μM) decreased forward scatter and increased annexin-V-binding significantly, events paralleled by increased [Ca(2+)]i and ceramide formation. Exposure to 1 was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) slightly, but significantly blunted the effect of 1 (2 μM) on annexin-V-binding. The present observations demonstrate that 1 may trigger suicidal death of erythrocytes, cells devoid of mitochondria and nuclei.

Topics: Aniline Compounds; Annexin A5; Apoptosis; Calcium; Cell Death; Cell Membrane; Cell Size; Ceramides; Cytosol; Erythrocytes; Hemolysis; Molecular Structure; Naphthoquinones; NF-kappa B; Phosphatidylserines; Vitamin K 3; Xanthenes

The possibility of newer allergens being responsible for atopy needs to be explored at regional level due to environmental variables. Current studies were undertaken to identify common environmental allergens causing atopy in a defined population of India and to correlate the presence of various risk factors with the clinical presentation of allergy. Newer allergens like human dander and rice grain dust were identified and reported as the most common cause of atopy in this region. Atopy, elevated serum total IgE and familial tendency, was observed in 88%, 69% and 58% of allergic patients respectively. Further, allergen-specific immune responses like lymphocyte proliferation and cytokine secretion were studied in vitro using peripheral blood mononuclear cells (PBMC) isolated from both allergic and non-allergic individuals. Although, some allergens induced significant lymphocyte proliferation in vitro, allergen-induced cytokine secretion except that of TNF-α was not seen. Significantly higher ratio of secreted IL-4/IFN-γ cytokines was observed in PBMC isolated from allergic subjects in response to PHA. Plumbagin (vitamin K3 analogue) completely inhibited PHA-induced cytokine production in PBMC, in both allergic and non-allergic individuals. Plumbagin modulated the levels of intracellular reactive oxygen species and glutathione and suppressed PHA induced activation of NF-κB in human PBMC. The results thus show in human PMBC, for the first time, the anti-allergic and anti-inflammatory effects of plumbagin and underscore its therapeutic potential.

Topics: Allergens; Blotting, Western; Cell Proliferation; Cytokines; Electrophoretic Mobility Shift Assay; Humans; Hypersensitivity, Immediate; Immunoglobulin E; Leukocytes, Mononuclear; Naphthoquinones; Reactive Oxygen Species; Surveys and Questionnaires; T-Lymphocytes; Vitamin K 3

Elicitation of Plumbago indica hairy roots with yeast carbohydrate fraction, chitosan, manganese chloride, copper chloride and methyl jasmonate exhibited significant elevation (~1.2 to 2 fold) of plumbagin production in shake flask culture as compared with control. Chitosan and methyl jasmonate elicitation also caused simultaneous plumbagin leaching into culture media. Three days' exposure of chitosan (200 mg l(-1)) and methyl jasmonate (80 μM) together synergized total plumbagin yield to its maximum 11.96 ± 0.76 mg g(-l) DW in shake flask culture. In bioreactor cultivation, a significant raise in fresh root biomass was recorded on day 20 as compared with control shake flask culture. Three days' exposure of chitosan (200 mg l(-1)) and methyl jasmonate (80 μM) with 20 days old bioreactor-culture significantly improved total plumbagin production to 13.16 ± 1.72 mg g(-l) DW with simultaneous plumbagin leaching into bioreactor media.

Topics: Acetates; Biomass; Bioreactors; Cell Culture Techniques; Cells, Cultured; Chitosan; Culture Media; Cyclopentanes; Fungi; Naphthoquinones; Oxylipins; Plant Roots; Plumbaginaceae

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, H-PLN) was isolated from Plumbago zeylanica, the anticancer traditional Chinese medicine (TCM). Five new lanthanide(III) complexes of deprotonated plumbagin: [Y(PLN)(3)(H(2)O)(2)] (1), [La(PLN)(3)(H(2)O)(2)] (2), [Sm(PLN)(3)(H(2)O)(2)]⋅H(2)O (3), [Gd(PLN)(3)(H(2)O)(2)] (4), and [Dy(PLN)(3)(H(2)O)(2)] (5) were synthesized by the reaction of plumbagin with the corresponding lanthanide salts, in amounts equal to ligand/metal molar ratio of 3:1. The PLN-lanthanide(III) complexes were characterized by different physicochemical methods: elemental analyses, UV-visible, IR and (1)H NMR and ESI-MS (electrospray ionization mass spectrum) as well as TGA (thermogravimetric analysis). The plumbagin and its lanthanide(III) complexes 1-5, were tested for their in vitro cytotoxicity against BEL7404 (liver cancer) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The five PLN-lanthanide (III) complexes 1-5 effectively inhibited BEL7404 cell lines growth with IC(50) values of 11.0±3.5, 5.1±1.3, 6.1±1.1, 6.4±1.3, and 9.8±1.5 μM, respectively, and exhibited a significantly enhanced cytotoxicity compared to plumbagin and the corresponding lanthanide salts, suggesting a synergistic effect upon plumbagin coordination to the Ln(III) ion. The lanthanide complexes under investigation also exerted dose- and time-dependent cytotoxic activity. [La(PLN)(3)(H(2)O)(2)] (2) and plumbagin interact with calf thymus DNA (ct-DNA) mainly via intercalation mode, but for [La(PLN)(3)(H(2)O)(2)] (2), the electrostatic interaction should not be excluded; the binding affinity of [La(PLN)(3)(H(2)O)(2)] (2) to DNA is stronger than that of free plumbagin, which may correlate with the enhanced cytotoxicity of the PLN-lanthanide(III) complexes.

Topics: Antineoplastic Agents; Cations; Cell Line, Tumor; DNA; Humans; Inhibitory Concentration 50; Lanthanoid Series Elements; Liver Neoplasms; Naphthoquinones; Organometallic Compounds; Pilot Projects; Spectrum Analysis; Thermogravimetry

A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 µg mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.

Topics: Animals; Bombyx; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Green Fluorescent Proteins; Larva; Naphthoquinones; Nucleopolyhedroviruses; Single-Chain Antibodies

In vitro biological activities including phytotoxic, antifungal activities as well as acute toxicity of the methanol extract, fractions and/or isolated compounds from the stem bark of Diospyros canaliculata were investigated. Well agar diffusion and macrodilution assays were used for investigating the antifungal activity. A phytotoxicity assay was performed against Lemna minor while an acute toxicity assay was performed in mice via oral administration. As a result, plumbagin (5-hydroxy-2-methyl-1,4-naphtoquinone) and two known pentacyclic triterpenes (lupeol and lupenone) were isolated from the extract. With regards the antifungal activities, the inhibition zones varied from 16.51 to 24.86 mm and from 20.50 to 25.10 mm for the extract and plumbagin, respectively. The minimum inhibitory concentrations of the extract and plumbagin ranged between 12.5-25 and 0.78-1.56 µg mL(-1), respectively. At 50 µg mL(-1), the hexane fraction showed phytotoxic activities similar to paraquat, the standard phytotoxic inhibitor. The extract was found to be non-toxic to mice after administration per os. Based on the current findings, we can conclude that this extract is non toxic, with significant phytotoxic and antifungal properties due to the presence of plumbagin.

Topics: Animals; Anti-Inflammatory Agents; Antifungal Agents; Araceae; Diospyros; Mice; Microbial Sensitivity Tests; Naphthoquinones; Paraquat; Pentacyclic Triterpenes; Plant Bark; Plant Extracts; Triterpenes

Plumbagin, a quinonoid found in the plants of the Plumbaginaceae, possesses medicinal properties. In this study we investigated the anti-proliferative and apoptotic activity of plumbagin by using two human colonic cancer cell lines, HT29 and HCT15. IC50 of Plumbagin for HCT15 and HT29 cells (22.5 µM and 62.5 µM, respectively) were significantly different. To study the response of cancer cells during treatment strategies, cells were treated with two different concentrations, 15 µM, 30 µM for HCT15 and 50 µM, 75 µM for HT29 cells. Though activation of NFκB, Caspases-3, elevated levels of TNF-α, cytosolic Cytochrome C were seen in both HCT15 cells HT29 treated with plumbagin, aberrant apoptosis with decreased level of pEGFR, pAkt, pGsk-3β, PCNA and Cyclin D1was observed only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells. This suggests that plumbagin induces apoptosis in both HCT15 cells and HT29 treated, whereas, proliferation was inhibited only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells, but not in 50 µM plumbagin treated HT29 cells. Expression of COX-2 was decreased in 75 µM plumbagin treated HT29 cells when compared to 50 µM plumbagin treated HT29 cells, whereas HCT15 cells lack COX. Hence the observed resistance to induction of apoptosis in 50 µM plumbagin treated HT29 cells are attributed to the expression of COX-2. In conclusion, plumbagin induces apoptosis in colonic cancer cells through TNF-α mediated pathway depending on expression of COX-2 expression.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Comet Assay; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Naphthoquinones; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Tetrazolium Salts; Thiazoles

Adverse reactions of acetaminophen have been associated to oxidative stress, which may be elicited by reactive oxygen species (ROS) and/or production of the metabolite NAPQI. Both phenomena would arise through the activity of liver cytochrome P450 (CYP450) system, but their contribution to this oxidative stress is yet to be clarified. A NADPH oxidase activity has been proposed in rat liver microsomes. This activity may be due to the presence of NAD(P)H oxidase (NOX) isoforms in liver endoplasmic reticulum. Both NOX and the CYP450 system activities can catalyze ROS generation using NADPH as a cofactor. Therefore, acetaminophen biotransformation, which requires NADPH, may promote ROS generation through either activity or both. To discriminate between these possibilities, rat liver microsomes were incubated with acetaminophen and NADPH in the presence or absence of specific inhibitors. Incubation with NADPH and acetaminophen elicited lipid peroxidation and decreased thiol content and glutathione-S-transferase (GST) activity. The NOX inhibitors apocynin and plumbagin prevented all these phenomena but the decrease in thiol content. In contrast, this decrease was completely prevented by the specific CYP450 system inhibitor SKF-525A. These data suggest that ROS generation following incubation of microsomes with acetaminophen and NADPH appears to be mainly caused by a NOX activity. In light of these data, toxicity of acetaminophen is discussed.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Antipyretics; Catechin; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Interactions; Glutathione Transferase; Lipid Peroxidation; Male; Microsomes, Liver; NADP; Naphthoquinones; Oxidative Stress; Proadifen; Rats; Rats, Sprague-Dawley

This study examined the ability of plumbagin to induce apoptosis in chronic myelogenous leukemia (CML). Plumbagin exposure led to a significant reduction in cell viability and the induction of apoptosis. Mechanistically, plumbagin treatment led to elevated levels of ROS. Plumbagin-induced apoptosis was inhibited by N-acetyl L-cysteine (NAC) and PEG-catalase. Furthermore, plumbagin exposure led to elevated expression of DR4 and DR5 and increased killing through soluble TRAIL. The plumbagin-induced increase in DR4 and DR5 was inhibited by treatment with NAC. Together, this study suggests that plumbagin may be an effective treatment of CML through increased sensitivity to TRAIL-mediated killing.

Topics: Acetylcysteine; Apoptosis; Blotting, Western; Catalase; Cell Survival; Flow Cytometry; Gene Expression; Humans; In Situ Nick-End Labeling; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Polyethylene Glycols; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction

Hormesis occurs when a low level stress elicits adaptive beneficial responses that protect against subsequent exposure to severe stress. Recent findings suggest that mild oxidative and thermal stress can extend lifespan by hormetic mechanisms. Here we show that the botanical pesticide plumbagin, while toxic to C. elegans nematodes at high doses, extends lifespan at low doses. Because plumbagin is a naphthoquinone that can generate free radicals in vivo, we investigated whether it extends lifespan by activating an adaptive cellular stress response pathway. The C. elegans cap'n'collar (CNC) transcription factor, SKN-1, mediates protective responses to oxidative stress. Genetic analysis showed that skn-1 activity is required for lifespan extension by low-dose plumbagin in C. elegans. Further screening of a series of plumbagin analogs identified three additional naphthoquinones that could induce SKN-1 targets in C. elegans. Naphthazarin showed skn-1dependent lifespan extension, over an extended dose range compared to plumbagin, while the other naphthoquinones, oxoline and menadione, had differing effects on C. elegans survival and failed to activate ARE reporter expression in cultured mammalian cells. Our findings reveal the potential for low doses of naturally occurring naphthoquinones to extend lifespan by engaging a specific adaptive cellular stress response pathway.

Topics: Aging; Animals; Biosensing Techniques; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Hep G2 Cells; Humans; Longevity; Mutation; Naphthoquinones; Stress, Physiological; Survival Analysis; Tetrahydronaphthalenes; Toxins, Biological; Transcription Factors; Transcription, Genetic; Vitamin K 3

Prey capture and digestion in Nepenthes spp. through their leaf-evolved biological traps involve a sequence of exciting events. Sugar-rich nectar, aroma chemicals, narcotic alkaloid secretions, slippery wax crystals, and other biochemicals take part in attracting, capturing, and digesting preys in Nepenthes pitchers. Here we report the distribution of three potent naphthoquinones in Nepenthes khasiana and their roles in prey capture. Plumbagin was first detected in N. khasiana, and its content (root: 1.33 ± 0.02%, dry wt.) was the highest found in any natural source. Chitin induction enhanced plumbagin levels in N. khasiana (root: 2.17 ± 0.02%, dry wt.). Potted N. khasiana plants with limited growth of roots and aerial parts, showed higher levels of plumbagin accumulation (root: 1.92 ± 0.02%; root, chitin induction: 3.30 ± 0.21%, dry wt.) compared with field plants. Plumbagin, a known toxin, insect ecdysis inhibitor, and antimicrobial, was also found embedded in the waxy layers at the top prey capture region of N. khasiana pitchers. Chitin induction, mimicking prey capture, produced droserone and 5-O-methyl droserone in N. khasiana pitcher fluid. Both these naphthoquinone derivatives provide antimicrobial protection to the pitcher fluid from visiting preys. A two-way barrier was found between plumbagin and its two derivatives. Plumbagin was never detected in the pitcher fluid whereas both its derivatives were only found in the pitcher fluid on chitin induction or prey capture. The three naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone, act as molecular triggers in prey capture and digestion in the carnivorous plant, N. khasiana.

Topics: Animals; Chitin; Chromatography, High Pressure Liquid; Insecta; Magnoliopsida; Naphthoquinones; Predatory Behavior

Increasing evidence indicates that the interaction between the CXC chemokine receptor-4 (CXCR4) and its ligand CXCL12 is critical in the process of metastasis that accounts for more than 90% of cancer-related deaths. Thus, novel agents that can downregulate the CXCR4/CXCL12 axis have therapeutic potential in inhibiting cancer metastasis.. In this report, we investigated the potential of an agent, plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), for its ability to modulate CXCR4 expression and function in various tumor cells using Western blot analysis, DNA binding assay, transient transfection, real time PCR analysis, chromatin immunoprecipitation, and cellular migration and invasion assays.. We found that plumbagin downregulated the expression of CXCR4 in breast cancer cells irrespective of their HER2 status. The decrease in CXCR4 expression induced by plumbagin was not cell type-specific as the inhibition also occurred in gastric, lung, renal, oral, and hepatocellular tumor cell lines. Neither proteasome inhibition nor lysosomal stabilization had any effect on plumbagin-induced decrease in CXCR4 expression. Detailed study of the underlying molecular mechanism(s) revealed that the regulation of the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression, inhibition of NF-κB activation, and suppression of chromatin immunoprecipitation activity. In addition, using a virtual, predictive, functional proteomics-based tumor pathway platform, we tested the hypothesis that NF-κB inhibition by plumbagin causes the decrease in CXCR4 and other metastatic genes. Suppression of CXCR4 expression by plumbagin was found to correlate with the inhibition of CXCL12-induced migration and invasion of both breast and gastric cancer cells.. Overall, our results indicate, for the first time, that plumbagin is a novel blocker of CXCR4 expression and thus has the potential to suppress metastasis of cancer.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Computer Simulation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Models, Biological; Naphthoquinones; Neoplasm Invasiveness; NF-kappa B; Protein Binding; Receptors, CXCR4; Stomach Neoplasms; Transcription, Genetic

Successful embryogenesis is a critical rate limiting step for the survival and transmission of parasitic worms as well as pathology mediated by them. Hence, blockage of this important process through therapeutic induction of apoptosis in their embryonic stages offers promise for developing effective anti-parasitic measures against these extra cellular parasites. However, unlike in the case of protozoan parasites, induction of apoptosis as a therapeutic approach is yet to be explored against metazoan helminth parasites.. For the first time, here we developed and evaluated flow cytometry based assays to assess several conserved features of apoptosis in developing embryos of a pathogenic filarial nematode Setaria digitata, in-vitro as well as ex-vivo. We validated programmed cell death in developing embryos by using immuno-fluorescence microscopy and scoring expression profile of nematode specific proteins related to apoptosis [e.g. CED-3, CED-4 and CED-9]. Mechanistically, apoptotic death of embryonic stages was found to be a caspase dependent phenomenon mediated primarily through induction of intracellular ROS. The apoptogenicity of some pharmacological compounds viz. DEC, Chloroquine, Primaquine and Curcumin were also evaluated. Curcumin was found to be the most effective pharmacological agent followed by Primaquine while Chloroquine displayed minimal effect and DEC had no demonstrable effect. Further, demonstration of induction of apoptosis in embryonic stages by lipid peroxidation products [molecules commonly associated with inflammatory responses in filarial disease] and demonstration of in-situ apoptosis of developing embryos in adult parasites in a natural bovine model of filariasis have offered a framework to understand anti-fecundity host immunity operational against parasitic helminths.. Our observations have revealed for the first time, that induction of apoptosis in developing embryos can be a potential approach for therapeutic intervention against pathogenic nematodes and flow cytometry can be used to address different issues of biological importance during embryogenesis of parasitic worms.

Topics: Animals; Antinematodal Agents; Apoptosis; Caenorhabditis elegans Proteins; Calcium-Binding Proteins; Caspases; Cattle; Cell Membrane; Chloroquine; Curcumin; Cytochromes c; Cytoplasm; Embryo, Nonmammalian; Female; Flow Cytometry; Lipid Peroxidation; Microscopy, Fluorescence; Naphthoquinones; Primaquine; Reactive Oxygen Species; Setaria Nematode

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

Plumbagin (PL), a herbal compound derived from roots of the medicinal plant Plumbago zeylanica, has been shown to have immunosuppressive properties. Present report describes that PL is a potent novel agent in control of encephalitogenic T cell responses and amelioration of mouse experimental autoimmune encephalomyelitis (EAE), through down-regulation of JAK-STAT pathway. PL was found to selectively inhibit IFN-γ and IL-17 production by CD4(+) T cells, which was mediated through abrogated phosphorylation of JAK1 and JAK2. Consistent with IFN-γ and IL-17 reduction was suppressed STAT1/STAT4/T-bet pathway which is critical for Th1 differentiation, as well as STAT3/ROR pathway which is essential for Th17 differentiation. In addition, PL suppressed pro-inflammatory molecules such as iNOS, IFN-γ and IL-6, accompanied by inhibition of IκB degradation as well as NF-κB phosphorylation. These data give new insight into the novel immune regulatory mechanism of PL and highlight the great value of this kind of herb compounds in probing the complex cytokine signaling network and novel therapeutic targets for autoimmune diseases.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cytokines; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Female; Immunohistochemistry; Inflammation Mediators; Janus Kinases; Mice; Mice, Inbred C57BL; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Naphthoquinones; NF-kappa B; Signal Transduction; Spinal Cord; STAT Transcription Factors; T-Lymphocytes

Several reports have demonstrated the anticancer activities of plumbagin, a naphthoquinone derivative isolated from plants belonging to Plumbaginaceae family. However, to the best of our knowledge, there are no reports which describe gold nanoconjugation with plumbagin, even though plumbagin is considered to be a promising therapeutic agent. In this report, we demonstrate the fabrication and characterization of gold nanoparticles conjugated with plumbagin (AuPB) that can reduce the toxicity of the latter, and their capacity for cellular localization and generation of reactive oxygen species. The anticancer activity and ability of plumbagin to produce reactive oxygen species was studied and compared with that of bromoderivatives of 1,4 naphthoquinones such as 2-bromo-1,4-naphthoquinone (2-BNQ) and 2,3-dibromo-1, 4-naphthoquinone (2,3-DBNQ) and their gold nanoconjugates. Plumbagin and bromoderivatives of 1,4 naphthoquinones in the form of gold nanoconjugates showed reduced cytotoxicity and apoptosis compared with the pristine compounds, ie, plumbagin, 2-BNQ, and 2,3-DBNQ. Interestingly, we observed that the gold nanoparticles could quench the reactive oxygen species-generating capacity of plumbagin, 2-BNQ, and 2,3-BNQ, which is one of the main mechanisms of action of the naphthoquinones. Therefore, it can be concluded that conjugation with gold nanoparticles can reduce the cytotoxicity of these compounds.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Fluoresceins; Gold; Histocytochemistry; Humans; Metal Nanoparticles; Naphthoquinones; Reactive Oxygen Species; Tumor Suppressor Protein p53

Plumbago scandens L. is a Brazilian tropical/subtropical species that occurs along the coast. Chemically it is mainly represented by naphthoquinones, flavonoids, terpenoids and steroids. The aim of the present work is to study quantitative changes in the root metabolic production of Plumbago scandens during different physiologic developmental stages relative to floration. The results indicated the presence of four substances in the extracts: plumbagin, epi-isoshinanolone, palmitic acid and sitosterol, independent on developmental stage. The naphthoquinone plumbagin has always showed to be the major component of all extracts. Naphthoquinones exhibited their highest content during floration, while the content of the two others components decreased during this stage, revealing an inverse profile. The chemical composition changed depending on the plant requirements.

Topics: Chromatography, Gas; Naphthoquinones; Palmitic Acid; Plant Roots; Plumbaginaceae; Sitosterols; Tetrahydronaphthalenes

Plumbagin, a naphtoquinone from the roots of Plumbago zeylanica is known to possess anticancer and anti-bacterial activity. Based on the former finding of our group in vitro demonstrating its effectiveness in human promyelocytic leukemia cells, NB4, in this study we further revealed the mitochondrial pathway involved in plumbagin-induced apoptosis. We also found that the generation of ROS was a critical mediator in plumbagin-induced apoptosis, which would be abrogated completely by antioxidant, NAC. The anticancer effect of plumbagin was investigated in vivo using NB4 tumor xenograft in NOD/SCID mice. The incidence of formation, growth characteristics, body weight and volume of tumors were observed. The histopathologic examination of tumors and organs were made. The results showed that intraperitoneal injection of plumbagin (2mg/kg body weight) daily for 3 weeks resulted to a 64.49% reduction of tumor volume compared with the control. Furthermore, there was no overt manifestation of toxicity such as weight loss, tissue damage and behavior change which appeared in Doxorubicin-treated mice (1mg/kg thrice a week). These results indicate that plumbagin has potential as a novel therapeutic agent for myeloid leukemia.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Enzyme Activation; Humans; In Situ Nick-End Labeling; Leukemia, Promyelocytic, Acute; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred NOD; Mice, SCID; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Xenograft Model Antitumor Assays

This study first investigates the anti-metastatic effect of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMPs and u-PA expressions in human lung cancer cells, A549. First, the result demonstrated plumbagin could inhibit TPA induced the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay and Boyden chamber assay. Data also showed plumbagin could inhibit the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) involved in the down-regulating enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and urokinase-type plasminogen activator (u-PA) induced by TPA. Next, plumbagin also strongly inhibited TPA-induced phosphorylation and degradation of inhibitor of kappaBalpha (IkappaBalpha), and the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Also, a dose-dependent inhibition on the binding abilities of NF-kappaB and activator protein-1 (AP-1) by plumbagin treatment was further observed. Further, the treatment of specific inhibitor for ERK (U0126) to A549 cells could inhibit TPA-induced MMP-2 and u-PA expressions along with an inhibition on cell invasion and migration. Presented data reveals that plumbagin is a novel, effective, anti-metastatic agent that functions by down-regulating MMP-2 and u-PA gene expressions.

Topics: Cell Line, Tumor; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Mitogen-Activated Protein Kinase 3; Naphthoquinones; NF-kappa B; Phosphorylation; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Urokinase-Type Plasminogen Activator

Many phytochemicals function as noxious agents that protect plants against insects and other damaging organisms. However, at subtoxic doses, the same phytochemicals may activate adaptive cellular stress response pathways that can protect cells against a variety of adverse conditions. We screened a panel of botanical pesticides using cultured human and rodent neuronal cell models, and identified plumbagin as a novel potent activator of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. In vitro, plumbagin increases nuclear localization and transcriptional activity of Nrf2, and induces the expression of the Nrf2/ARE-dependent genes, such as heme oxygenase 1 in human neuroblastoma cells. Plumbagin specifically activates the Nrf2/ARE pathway in primary mixed cultures from ARE-human placental alkaline phosphatase reporter mice. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provides protection against subsequent oxidative and metabolic insults. The neuroprotective effects of plumbagin are abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo, administration of plumbagin significantly reduces the amount of brain damage and ameliorates-associated neurological deficits in a mouse model of focal ischemic stroke. Our findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cerebral Cortex; Cerebral Infarction; Disease Models, Animal; Embryo, Mammalian; Gene Expression Regulation; Glucose; Heme Oxygenase-1; Humans; Hypoxia; Infarction, Middle Cerebral Artery; Mice; Mice, Inbred C57BL; Naphthoquinones; Neuroblastoma; Neurologic Examination; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; Rats; Rats, Sprague-Dawley; Transcription Factor AP-1; Transfection

The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.

Topics: Cell Line, Tumor; Cell Nucleus; DNA; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Enzyme Induction; Humans; Models, Biological; Naphthoquinones; Phosphorylation; Protein Binding; Protein Transport; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Signal Transduction; STAT3 Transcription Factor; Vitamin K 3

Glucose 6-phosphate (G6P) is a metabolic intermediate with many possible cellular fates. In mycobacteria, G6P is a substrate for an enzyme, F(420)-dependent glucose-6-phosphate dehydrogenase (Fgd), found in few bacterial genera. Intracellular G6P levels in six Mycobacterium sp. were remarkably higher ( approximately 17-130-fold) than Escherichia coli and Bacillus megaterium. The high G6P level in Mycobacterium smegmatis may result from 10-25-fold higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase when grown on glucose, glycerol, or acetate compared with B. megaterium and E. coli. In M. smegmatis this coincided with up-regulation of the first gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, when acetate was the carbon source, suggesting a cellular program for maintaining high G6P levels. G6P was depleted in cells under oxidative stress induced by redox cycling agents plumbagin and menadione, whereas an fgd mutant of M. smegmatis used G6P less well under such conditions. The fgd mutant was more sensitive to these agents and, in contrast to wild type, was defective in its ability to reduce extracellular plumbagin and menadione. These data suggest that intracellular G6P in mycobacteria serves as a source of reducing power and, with the mycobacteria-specific Fgd-F(420) system, plays a protective role against oxidative stress.

Topics: Antioxidants; Bacillus megaterium; Cell Membrane; Escherichia coli; Genetic Complementation Test; Glucose-6-Phosphate; Models, Biological; Mutation; Mycobacterium; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Riboflavin; Vitamin K 3

The present study was aimed to evaluate the anti-tumor efficacy and systemic toxicity of chitosan-based plumbagin microspheres in comparison to free plumbagin. The optimized formulation had a mean particle size of 106.35 mum with an encapsulation efficiency of 80.12%. Pharmacokinetic studies showed a 22.2-fold increase in elimination half-life (t(1/2)) of plumbagin from chitosan microspheres as compared to free plumbagin. Administration of plumbagin microspheres resulted in a significant tumor growth inhibition and reduced systemic toxicity. These results suggest that chitosan-based microspheres could be a promising strategy for the systemic delivery of anti-cancer agents like plumbagin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Blood Cell Count; Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Chitosan; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Delayed-Action Preparations; Drug Compounding; Glutaral; Half-Life; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Microspheres; Naphthoquinones; Particle Size; Spectrophotometry, Ultraviolet

Methyl jasmonate, 50 microM, 0.5 mg yeast extract/l and 100 mg chitosan/l stimulated plumbagin production in Drosera burmanii whole plant cultures after 6 days of elicitation. Yeast extract (0.5 mg/l) was the most efficient enhancing plumbagin production in roots of D. burmanii to 8.8 +/- 0.5 mg/g dry wt that was 3.5-fold higher than control plants.

Topics: Cells, Cultured; Culture Media; Drosera; Humans; Naphthoquinones

A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly(4)Ser)(2) between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL(-1)) was improved to eightfold higher than that in conventional ELISA (0.2 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.

Topics: Cross Reactions; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Fluorescent Antibody Technique, Direct; Immunosorbents; Limit of Detection; Naphthoquinones; Single-Chain Antibodies

N-Acetylglucosaminylinositol (GlcNAc-Ins)-deacetylase (MshB) and mycothiol-S-conjugate amidase (Mca), structurally related amidases present in mycobacteria and other Actinomycetes, are involved in the biosynthesis of mycothiol and in the detoxification of xenobiotics as their mycothiol-S-conjugates, respectively. With substrate analogs of GlcNAc-Ins, MshB showed a marked preference for inositol as the aglycon present in GlcNAc-Ins. The inhibition of MshB and Mca by 10 thioglycosides, 7 cyclohexyl-2-deoxy-2-C-alkylglucosides, and 4 redox cyclers was evaluated. The latter contained plumbagin tethered via 2 to 5 methylene carbons and an amide linkage to phenyl-2-deoxy-2-amino-1-thio-alpha-d-glucopyranoside. These proved to be the most potent amongst the 21 compounds tested as inhibitors of MshB. Their inhibitory potency varied with the length of the spacer, with the compound with longest spacer being the most effective.

Topics: Acetylcysteine; Amidohydrolases; Bacterial Proteins; Cell Survival; Cysteine; Enzyme Inhibitors; Glycopeptides; Indicators and Reagents; Inositol; Mycobacterium tuberculosis; NADH, NADPH Oxidoreductases; Naphthoquinones; Oxidation-Reduction; Structure-Activity Relationship; Substrate Specificity; Thioglucosides

Plumbagin, derived from the plant Plumbago zeylanica, has been shown to chronically activate ERK1/2 and inhibit Akt activity in cancer cells. However, the acute effects of plumbagin on ERK1/2 and Akt activities remain unknown. In this study, we examined the effects of plumbagin on ERK1/2 and Akt activities in 3T3-L1 cells. Exposure of 3T3-L1 cells to plumbagin generated superoxide and activated both ERK1/2 and Akt. The plumbagin-stimulated ERK1/2 and Akt activities were sensitive to an antioxidant NAC, superoxide dismutase mimetic MnTBAP, superoxide scavenger Tiron and NAD(P)H oxidase inhibitor DPI. Plumbagin-stimulated ERK1/2 activity was attenuated by the MEK1/2 inhibitor PD98059 and Ras inhibitor manumycin A, whereas plumbagin-stimulated Akt activity was blocked by the PI3K inhibitor LY294002. Both plumbagin-stimulated ERK1/2 and Akt activities were attenuated by PP2, a Src inhibitor. Interestingly, inhibition of phosphatidylinositol 3-kinase (PI3-kinase), but not Akt, activity leaded to attenuation of plumbagin-stimulated ERK1/2 activity. These results suggest that plumbagin activates NAD(P)H oxidase, Src, and PI3K, and that the activated PI3K or PDK1 subsequently stimulate Akt and Ras-Raf-MEK1/2-ERK1/2 in 3T3-L1 cells.

Topics: 3T3-L1 Cells; Animals; Antineoplastic Agents, Phytogenic; Chromones; Drug Interactions; Enzyme Activation; Flavonoids; Free Radical Scavengers; Mice; Mitogen-Activated Protein Kinase 1; Morpholines; Naphthoquinones; Phosphoinositide-3 Kinase Inhibitors; Polyenes; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Pyrimidines; src-Family Kinases; Superoxides

Plumbagin (5-hydroxy-2-methyl-1,4 naphthoquinone) is a naturally occurring low molecular weight lipophilic phytochemical derived from roots of plants of the Plumbago genus. Plumbagin has been reported to have several clinically relevant biological activities in non-neural cells, including anti-atherosclerotic, anticoagulant, anticarcinogenic, antitumor, and bactericidal effects. In a recent screen of a panel of botanical pesticides, we identified plumbagin as having neuroprotective activity. In this study, we determined if plumbagin could modify the developmental fate of rat E14.5 embryonic neural progenitor cells (NPC). Plumbagin exhibited no cytotoxicity when applied to cultured NPC at concentrations below 1 μM. At a concentration of 0.1 μM, plumbagin significantly enhanced the proliferation of NPC as indicated by a 17% increase in the percentage of cells incorporating bromo-deoxyuridine. Plumbagin at a concentration of 0.1 pM (but not 0.1 μM), stimulated the production of astrocytes as indicated by increased GFAP expression. Plumbagin selectively induced the proliferation and differentiation of glial progenitor cells without affecting the proliferation or differentiation of neuron-restricted progenitors. Plumbagin (0.1 pM) rapidly activated the transcription factor signal transducer and activator of transcription 3 (Stat3) in NPC, and a Stat3 inhibitor peptide prevented both plumbagin-induced astrocyte formation and proliferation. These findings demonstrate the ability of a low molecular weight naturally occurring phytochemical to control the fate of glial progenitor cells by a mechanism involving the Stat3 signaling pathway.

Topics: Animals; Astrocytes; Cell Survival; Embryonic Stem Cells; Female; Naphthoquinones; Neurons; Neuroprotective Agents; Pregnancy; Rats; Rats, Sprague-Dawley; Spinal Cord; STAT3 Transcription Factor; Stem Cells

Electron transport and respiratory pathways are active in both latent and rapidly growing mycobacteria and remain conserved in all mycobacterial species. In mycobacteria, menaquinone is the sole electron carrier responsible for electron transport. Menaquinone biosynthesis pathway is found to be essential for the growth of mycobacteria. Structural analogs of the substrate or product of this pathway are found to be inhibitory for the growth of Mycobacterium smegmatis and M. tuberculosis. Several plumbagin [5-hydroxy-2-methyl-1, 4-naphthaquinone] derivatives have been analyzed for their inhibitory effects of which butyrate plumbagin was found to be most effective on M. smegmatis mc(2)155, whereas crotonate plumbagin showed greater activity on M. tuberculosis H37Rv. Effect on electron transport and respiration was demonstrated by butyrate plumbagin inhibiting oxygen consumption in M. smegmatis. Structural modifications of these molecules can further be improved upon to generate new molecules against mycobacteria.

Topics: Antitubercular Agents; Microbial Sensitivity Tests; Mycobacterium; Mycobacterium Infections; Mycobacterium smegmatis; Mycobacterium tuberculosis; Naphthoquinones

Plumbagin inhibited activation, proliferation, cytokine production, and graft-versus-host disease in lymphocytes and inhibited growth of tumor cells by suppressing nuclear factor-kappaB (NF-kappaB). Plumbagin was also shown to induce reactive oxygen species (ROS) generation in tumor cells via an unknown mechanism. Present report describes a novel role of cellular redox in modulation of immune responses in normal lymphocytes by plumbagin. Plumbagin depleted glutathione (GSH) levels that led to increase in ROS generation. The decrease in GSH levels was due to direct reaction of plumbagin with GSH as evinced by mass spectrometric and HPLC analysis. Further, addition of plumbagin to cells resulted in decrease in free thiol groups on proteins and increase in glutathionylation of proteins. The suppression of mitogen-induced T-cell proliferation and cytokine (IL-2/IL-4/IL-6/IFN-gamma) production by plumbagin was abrogated by thiol antioxidants but not by non-thiol antioxidants confirming that thiols but not ROS play an important role in biological activity of plumbagin. Plumbagin also abrogated mitogen-induced phosphorylation of ERK, IKK, and degradation of IkappaB-alpha. However, it did not affect phosphorylation of P38, JNK, and AKT. Our results for the first time show that antiproliferative effects of plumbagin are mediated by modulation of cellular redox. These results provide a rationale for application of thiol-depleting agents as anti-inflammatory drugs.

Topics: Adjuvants, Immunologic; Animals; Blotting, Western; Catalase; Cell Proliferation; Cells, Cultured; Concanavalin A; Cytokines; Glutathione; Inflammation Mediators; Interferon-gamma; Interleukin-2; Interleukin-4; Interleukin-6; Intracellular Space; Lymphocyte Activation; Male; Mice; Mitogens; Naphthoquinones; Oxidation-Reduction; Reactive Oxygen Species; Sulfhydryl Compounds; Superoxide Dismutase; T-Lymphocytes

Extracts from plants containing plumbagin (PLB) continue to be used as a treatment of a number of chronic immunologically-based diseases. However, most of these claims are supported only by anecdotal evidence with few scientific reports describing the mechanism of action or the efficacy of plumbagin in the suppression of the immune response. In the current study, we tested the hypothesis that plumbagin-induced suppression of the immune response was mediated through the induction of apoptosis. Splenocytes from C57BL/6 mice cultured in the presence of 0.5 microM or greater concentrations of PLB significantly reduced proliferative responses to mitogens, including anti-CD3 mAbs, concanavalin A (Con A), lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) in vitro. Exposure of naïve and activated splenocytes to PLB led to a significant increase in the levels of apoptosis. In addition, PLB treatment led to a significant increase in the levels of reactive oxygen species (ROS) in naïve and activated splenocytes. Furthermore, treatment with the ROS scavenger, N-acetylcysteine (NAC), prevented PLB-induced apoptosis, suggesting a role of ROS in PLB-induced apoptosis. PLB-induced apoptosis led to ROS-mediated activation of both the extrinsic and intrinsic apoptotic pathways. In addition, plumbagin led to increased expression of Fas. Finally, treatment of mice with PLB (5mg/kg) led to thymic and splenic atrophy as well as a significant suppression of the response to SEB and dinitrofluorobenzene (DNFB) in vivo. Together, these results suggest that plumbagin has significant immunosuppressive properties which are mediated by generation of ROS, upregulation of Fas, and the induction of apoptosis.

Topics: Acetylcysteine; Animals; Apoptosis; Atrophy; Caspases; Dinitrofluorobenzene; Enterotoxins; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; fas Receptor; Female; Immunosuppressive Agents; JNK Mitogen-Activated Protein Kinases; Lymphocytes; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Spleen; Thymus Gland; Up-Regulation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. However, emergence of drug resistance limits its potential use. Plumbagin is a natural quinonoid compound isolated from plant. In this study, induced apoptosis effect of the combined treatment with plumbagin and TRAIL on human melanoma A375 cell line was examined and possible mechanism was investigated. The cells were divided into four groups: control group, plumbagin group (plumbagin, 5 or 10 mumol/L), TRAIL group (TRAIL, 30 ng/mL) and plumbagin+TRAIL group (combined treatment group). The apoptosis, and the expression of DR4 and DR5 were detected by flow cytometry. The activities of caspase-8 and caspase-3 were determined by colorimetric assay. The results showed that the apoptosis rate was 8.3% in TRAIL group, 10.35%-16.94% in plumbagin group and 52.39%-65.39% in combined treatment group, respectively, with the difference being significant between combined treatment group and plumbagin or TRAIL group (P<0.05 for each). Moreover, plumbagin alone could markedly up-regulate DR5 mRNA and protein expression, and slightly increase DR4 mRNA and protein expression. Treatment of human melanoma A375 cells with plumbagin resulted in the activation of Caspase-3, but not Caspase-8. These results suggest that plumbagin might be useful for TRAIL-based treatment for melanoma.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 8; Cell Line, Tumor; Humans; Melanoma; Naphthoquinones; Receptors, TNF-Related Apoptosis-Inducing Ligand; Skin Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

The roots of Plumbago zeylanica Linn. (Plumbaginaceae) are reputed to have a wide spectrum of therapeutic properties in the Ayurvedic system of medicine. They are useful in curing many ailments such as skin diseases, diarrhea, plague and leprosy.. The study was aimed at isolating, separating and evaluating the antimicrobial properties of compounds such as neoisoshinanolone and 1-epineo-isoshinanolone from the roots of P. zeylanica.. The crude petroleum ether extract of roots of P. zeylanica was subjected to repeated chromatographic techniques to separate compounds 2 and 3 along with plumbagin. Structure elucidation was carried out using nuclear magnetic resonance (NMR), infra red (IR) and mass spectroscopy. The serial dilution method was used to test antimicrobial activities and their minimum inhibitory concentration (MIC) expressed in microg/mL.. 1-Epineo-isoshinanolone is more active with a MIC of 12.5-25 microg/mL whereas neoisoshinanolone has recorded a MIC of 50-100 microg/mL. The activities are compared with plumbagin (0.78-3.13 microg/mL) and standards streptomycin for bacteria and nystatin for fungi.. Earlier researchers have established the presence of plumbagin in the roots of P. zeylanica and its antimicrobial activities. The structure elucidation of two more biologically active biogenetic precursors along with their activities in the root extracts has been established for the first time in the present study.. The root extract of P. zeylanica possesses good antimicrobial activity, which suggests its therapeutic use in the Ayurvedic system of medicine to cure skin diseases.

Topics: Anti-Infective Agents; Drug Discovery; Fungi; Gram-Negative Bacteria; Gram-Positive Bacteria; Isomerism; Magnetic Resonance Spectroscopy; Mass Spectrometry; Medicine, Ayurvedic; Microbial Sensitivity Tests; Molecular Structure; Naphthoquinones; Plant Extracts; Plant Roots; Plumbaginaceae; Spectrophotometry, Infrared; Tetrahydronaphthalenes; Transition Temperature

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) (PL) is a naturally occurring yellow pigment found in the plants of the Plumbaginaceae, Droseraceae, Ancistrocladaceae, and Dioncophyllaceae families. It has been reported that PL exhibits anticarcinogenic, anti-inflammatory, and analgesic activities. However, the mechanism underlying its anti-inflammatory action remains unknown. In the current study, we investigated and characterized the anti-inflammatory and analgesic effects of PL orally administrated in a range of dosages from 5 to 20 mg/kg. We also examined the role of nuclear factor κB (NF-κB) and proinflammatory cytokines and mediators in this effect. The results showed that PL significantly and dose-dependently suppressed the paw edema of rats induced by carrageenan and various proinflammatory mediators, including histamine, serotonin, bradykinin, and prostaglandin E(2). PL reduced the number of writhing episodes of mice induced by the intraperitoneal injection of acetic acid, but it did not reduce the writhing episode numbers induced by MgSO(4) in mice or prolong the tail-flick reaction time of rats to noxious thermal pain. Mechanistic studies showed that PL effectively decreased the production of the proinflammatory cytokines interleukin 1β, interleukin 6, and tumor necrosis factor α. It also inhibited the expression of the proinflammatory mediators inducible nitric-oxide synthase and cyclooxygenase 2, whereas it did not inhibit the expression of cyclooxygenase 1. Further studies demonstrated that PL suppressed inhibitor of κBα phosphorylation and degradation, thus inhibiting the phosphorylation of the p65 subunit of NF-κB. This study suggests that PL has a potential to be developed into an anti-inflammatory agent for treating inflammatory diseases.

Topics: Abdominal Pain; Acetic Acid; Analgesics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bradykinin; Carrageenan; Cyclooxygenase 2; Dinoprostone; Edema; Foot; Gene Expression; Histamine; Hot Temperature; I-kappa B Proteins; Inflammation; Interleukin-1beta; Interleukin-6; Magnesium Sulfate; Male; Mice; Mice, Inbred ICR; Naphthoquinones; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; Pain Threshold; Phosphorylation; Rats; Rats, Sprague-Dawley; Serotonin; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation.

Topics: Amino Acid Sequence; Antineoplastic Agents, Phytogenic; Bacterial Proteins; Carrier Proteins; Escherichia coli; Escherichia coli Proteins; Genetic Complementation Test; Microbial Sensitivity Tests; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Naphthoquinones; Sequence Alignment; Superoxide Dismutase; Superoxides

Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunoglobulin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.

Topics: Animals; Baculoviridae; Blotting, Western; Cell Line; Chromatography, Affinity; Chromatography, Ion Exchange; Cross Reactions; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Genetic Vectors; Molecular Structure; Naphthoquinones; Single-Chain Antibodies; Spodoptera

Benjakul is a Thai traditional medicine preparation, used for balanced health. From selective interviews of folk doctors in southern Thailand, it was used as the adaptogen drug for cancer patients. In our previous study, the ethanolic extract of Benjakul preparation exhibited high cytotoxic activity against lung cancer cell lines (COR-L23). Piperine has been identified as the main compound in the extract. In addition, plumbagin was found as the most cytotoxic compound. In this study, a reversed-phase high performance liquid chromatography (HPLC) method for quality control such as chemical fingerprint, quantification and stability of the ethanolic extract of Benjakul preparation was developed. The reversed-phase HPLC was performed with a gradient mobile phase composed of water and acetronitrile, and peaks were detected at 256 nm. Based on validation results, this analytical method is precise, accurate and stable for quantitative determination of piperine and plumbagin which are cytotoxic compounds isolated from the ethanolic extract of Benjakul preparation. This method could be suitable for analysis of Benjakul extract.

Topics: Alkaloids; Benzodioxoles; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Drug Stability; Drugs, Chinese Herbal; Humans; Medicine, Traditional; Naphthoquinones; Pharmaceutical Preparations; Piperaceae; Piperidines; Plant Extracts; Plants, Medicinal; Plumbaginaceae; Polyunsaturated Alkamides; Quality Control; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Thailand; Zingiber officinale

The aim of the present study was to determine the effect of three strains of Agrobacterium rhizogenes (ATCC 15834, A4 and LBA 9402) and the nature of explants (leaf and stem) on hairy root induction, growth and plumbagin production in Plumbago indica. The first appearance of hairy roots, the transformation frequency, dry root biomass and plumbagin accumulation were found to be maximum in hairy roots induced in leaf explants infected with A. rhizogenes ATCC 15834 as compared with the other two bacterial strains. The hairy roots generated from stem explants infected with all three strains were not found to be productive in terms of the selected parameters. Finally, the insertion of the rolB gene of A. rhizogenes ATCC 15834 in hairy roots of P. indica derived from leaf explants was confirmed by PCR analysis.

Topics: Biomass; Naphthoquinones; Plant Roots; Plumbaginaceae; Rhizobium

Plumbagin, a naphtoquinone present in the roots of Plumbago zeylanica, has been reported to have many beneficial effects such as antibacterial, antifungal, anticancer, antimutagenic and antioxidant effects, but this compound has also been reported to have many side effects. Given the wide use of P. zeylanica in traditional medicine and the various potential therapeutic uses of plumbagin, the present study was carried out to further elucidate the potential genotoxicity and antigenotoxicity of plumbagin in mouse lymphoma L5178Y cells, using the comet assay. Without affecting the cell viability, plumbagin itself was found to induce significant DNA damage at concentrations as low as 0.25 ng/ml. When the cells were exposed to non-DNA damaging concentrations of plumbagin, together with NQNO (known to interact with DNA in many different ways) or catechol (known to induce oxidative DNA damage), plumbagin was found to significantly reduce the catechol-induced DNA damage, but to be without protective effect against the NQNO-induced damage. The fact that non-DNA damaging concentrations of plumbagin diminished the DNA damage induced by catechol, provides further support for the idea that plumbagin may act as an antioxidative agent at low concentrations.

Topics: Animals; Antimutagenic Agents; Antineoplastic Agents, Phytogenic; Catechols; Cell Line, Tumor; Cell Survival; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Drug Screening Assays, Antitumor; Hydroxyquinolines; Lymphoma; Mice; Naphthoquinones

The aim of this study was to evaluate the antimicrobial activity of Drosophyllum lusitanicum leaf extract against various yeasts and bacteria species, including both standard and clinically isolated strains. The extract exhibited strong antimicrobial activity against all the tested yeast strains with inhibition zones ranging 23.67-42.23 mm and with minimum inhibitory concentration (MIC) values ranging 31-63 microg L(-1). All the Gram-positive bacteria studied were inhibited by the extract, showing inhibition zones ranging 17.67-43.00 mm and MIC values comprising between 15.6 and 250 microg L(-1). In contrast, the growth of the tested Gram-negative bacteria was not significantly affected by the extract. Among the microorganisms tested, Staphylococcus epidermidis ATCC 12228 was the most sensitive, presenting the lowest MIC value (15.6 microg L(-1)), while Enterococcus faecalis ATCC 29212 was the most tolerant (250 microg L(-1)). The extract of D. lusitanicum was analysed by gas chromatography-mass spectrometry and the major constituent found was plumbagin.

Topics: Anti-Infective Agents; Gas Chromatography-Mass Spectrometry; Gram-Negative Bacteria; Gram-Positive Bacteria; Magnoliopsida; Microbial Sensitivity Tests; Naphthoquinones; Plant Extracts; Plant Leaves; Yeasts

We constructed a single-chain variable fragment (scFv) antibody against plumbagin (PL) with improved specific binding to PL. Variable heavy- and light-chain genes were cloned directly from the cDNA of hybridoma cell line 3A3 and assembled using the splice-overlap extension polymerase chain reaction (SOE-PCR) with specific primers including flexible peptide (Gly(4)Ser)(3) linker primers. The constructed scFv gene was ligated into the pET28a expression vector and transformed into Escherichia coli BL21 (DE3). The denatured protein expressed as inclusion bodies in E. coli was solubilized, purified, and refolded by a stepwise dialysis. Intriguingly, the refolded scFv against PL displayed higher PL-binding specificity than that of its parental monoclonal antibody, MAb 3A3, which suggests the possibility of improving the function by constructing the scFv antibody. These notable properties of the recombinant antibody against PL made it possible to develop an enzyme-linked immunosorbent assay (ELISA) for reliable determination of PL.

Topics: Amino Acid Sequence; Base Sequence; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Immunoglobulin Variable Region; Magnoliopsida; Molecular Sequence Data; Naphthoquinones; Plumbaginaceae; Protein Binding; Protein Folding; Recombinant Proteins

Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), a quinone isolated from the roots of Plumbago zeylanica was recently reported to suppress the activation of NF-kappaB in tumor cells. NF-kappaB, a ubiquitous transcription factor, plays a central role in regulating diverse processes in leukocytes like cellular proliferation, expression of immunoregulatory genes and apoptosis during innate and adaptive immune responses. Consequently, plumbagin might affect the biological functions of leukocytes participating in various immune responses. The present report describes novel immunomodulatory effects of plumbagin. Plumbagin inhibited T cell proliferation in response to polyclonal mitogen Concanavalin A (Con A) by blocking cell cycle progression. It also suppressed expression of early and late activation markers CD69 and CD25 respectively, in activated T cells. At these immunosuppressive doses (up to 5 microM), plumbagin did not reduce the viability of lymphocytes. Further, the inhibition of T cell proliferation by plumbagin was accompanied by a decrease in the levels of Con A induced IL-2, IL-4, IL-6 and IFN-gamma cytokines. Similar immunosuppressive effects of plumbagin on cytokine levels were seen in vivo. To characterize the mechanism of inhibitory action of plumbagin, the mitogen induced IkappaB-alpha degradation and nuclear translocation of NF-kappaB was studied in lymphocytes. Plumbagin completely inhibited Con A induced IkappaB-alpha degradation and NF-kappaB activation. Further, plumbagin prevented Graft Versus Host Disease-induced mortality in mice. To our knowledge this is the first report showing the immunomodulatory effects of plumbagin in lymphocytes via modulation of NF-kappaB activation.

Topics: Active Transport, Cell Nucleus; Animals; Antigens, CD; Antigens, Differentiation; Antigens, Differentiation, T-Lymphocyte; Cell Cycle; Cell Nucleus; Cell Proliferation; Cells, Cultured; Cytokines; Down-Regulation; Graft vs Host Disease; I-kappa B Proteins; Immunosuppression Therapy; Interleukin-2 Receptor alpha Subunit; Lectins, C-Type; Lymphocyte Activation; Male; Mice; Naphthoquinones; NF-kappa B; Plant Roots; Plumbaginaceae; T-Lymphocytes; Transcriptional Activation

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria that serves as a barrier against harmful molecules, including antibiotics. The waaYZ locus that encodes the LPS core biosynthetic function in Escherichia coli was found to be induced strongly by superoxide generators but not by H(2)O(2), ethanol, or heat shock. This induction was dependent on SoxRS, a superoxide and nitric oxide sensing system, through a soxbox in the waaY promoter that binds SoxS. A DeltawaaYZ mutant became more sensitive to some superoxide generators, and the activation of SoxR by these drugs became more sensitized in the mutant. Through phenotypic microarray analysis, we found that the mutant became sensitive to a wide variety of chemicals not restricted to oxidizing agents. We found that the mutant is under envelope stress and is altered in LPS composition, as monitored by the level of sigma(E) activation and changes in the electrophoretic mobility of LPS, respectively. waaY expression was also regulated by MarA (multiple-antibiotic resistance regulator), which shares a binding site (soxbox) with SoxS, and was induced by salicylate, a nonoxidative compound. These results demonstrate a novel way of protecting gram-negative bacteria against various compounds by modifying LPS, possibly through phosphorylation. Since either oxidant or nonoxidant compounds elicit resistance toward themselves and other toxic drugs, this mechanism could serve as an efficient way for pathogenic bacteria to enhance survival during antibiotic treatment within an oxidant-rich host immune environment.

Topics: Bacterial Proteins; Blotting, Northern; DNA-Binding Proteins; Drug Resistance, Multiple, Bacterial; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Hydrogen Peroxide; Lipopolysaccharides; Naphthoquinones; Paraquat; Promoter Regions, Genetic; Trans-Activators; Transcription Factors; Vitamin K 3

G1-4A, a polysaccharide from an Indian medicinal plant Tinospora cordifolia, was recently shown to protect mice against septic shock by modulating the proinflammatory cytokines. G1-4A also activated B cells polyclonally. The present report describes in detail the molecular events associated with G1-4A-induced immunomodulation in vitro and in vivo. G1-4A treatment led to an increase in the CD69 expression in lymphocytes. G1-4A-induced proliferation of B cells was completely inhibited by PI3K inhibitor Ly294002, mTOR inhibitor rapamycin and NF-kappaB inhibitor plumbagin. Akt, ERK and JNK were activated by G1-4A which finally resulted in the activation of IKK, degradation of IkappaB-alpha and translocation of NF-kappaB to the nucleus. Administration of G1-4A to mice led to splenomegaly and an increase in the numbers of T cells, B cells and macrophages. This increase in spleen cellularity was due to in vivo proliferation of lymphocytes and upregulation of anti-apoptotic genes. Anti-TLR4-MD2 complex antibody inhibited G1-4A-induced B cell proliferation and degradation of IkappaB-alpha suggesting that TLR-4 was a receptor for G1-4A on B cells. Activation of RAW 264.7 macrophages by G1-4A was found to be dependent on ERK and NF-kappaB-mediated signals. The phagocytosis index in peritoneal exudate cells (PEC) isolated from G1-4A treated mice was significantly higher as compared to that in PEC from control mice. G1-4A administration also increased the number of CD11b(+) cells in the PEC without an increase in the total number of PEC. Thus the present understanding of the molecular mechanism of action of G1-4A, a novel non-microbial TLR4 agonist, will pave the way for its application as an immunomodulator and adjuvant.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; B-Lymphocytes; Carrier Proteins; Cell Line; Cell Proliferation; Chromones; Enzyme Inhibitors; Flavonoids; Lectins, C-Type; Lymphocyte Activation; Macrophages; MAP Kinase Kinase Kinases; Mice; Morpholines; Naphthoquinones; NF-kappa B; Phagocytosis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Polysaccharides; Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Sirolimus; Splenomegaly; Tinospora; Toll-Like Receptor 4; TOR Serine-Threonine Kinases

During recovery from lymphopenia, the naïve T-cells undergo homeostasis driven proliferation (HDP) and acquire a memory phenotype. The HDP of T-cells requires signals derived from T-cell-receptor, p56lck kinase, IL-7R and IL-15R. However, the role of other signaling molecules during HDP of CD4+ T-cells remains speculative. The differentiation of naïve T-cells into Th1/Th2/Th17 or Treg populations during HDP is not well understood. Present report describes the spatial and signaling characteristics of HDP of CD4+ T-cells and their cytokine profiles. The HDP of CD4+ T-cells was found to occur only in specific areas (T-cell zones) of secondary lymphoid organs of lymphopenic mice. The inhibitors of MEK and PKC and their combination with inhibitors of PI3kinase and mTOR suppressed mitogen induced T-cell proliferation without affecting their HDP. The CD4+ T-cells taken from reconstituted lymphopenic mice showed activation of proteins involved in NF-kappaB pathway, significantly higher production of pro-inflammatory cytokine IL-6, and lower production of IL-4 as compared to T-cells from normal mice. Plumbagin, a known NF-kappaB blocker inhibited survival as well as HDP of CD4+ T-cells and IL-6 production in activated T-cells. Our results demonstrate the essential role of NF-kappaB during HDP of T-cells.

Topics: Animals; Ascitic Fluid; CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Cytokines; Homeostasis; Immunologic Memory; Kinetics; Lymph Nodes; Lymphopenia; MAP Kinase Kinase Kinases; Mice; Mice, Inbred BALB C; Naphthoquinones; NF-kappa B; Peritoneal Cavity; Protein Kinase C; Signal Transduction; Spleen

The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the -35 and -10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

Topics: Agrobacterium tumefaciens; Bacterial Proteins; Binding Sites; Copper; DNA Footprinting; DNA Transposable Elements; Gene Expression Regulation, Bacterial; Genome, Bacterial; Mutation; Naphthoquinones; Operon; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Trace Elements; Vitamin K 3

The aim of this study was to evaluate the antimycobacterial and antigonorrhoeal activities of three naphthoquinones (diospyrone, crassiflorone and plumbagin) from Diospyros canaliculata and Diospyros crassiflora as well as the crude extracts from these plants. The agar disk diffusion assay, broth microdilution method, microplate Alamar blue assay (MABA) and radiometric respiratory technique using the BACTEC 460 TB system were used. Results of the antimycobacterial assays indicated that the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations ranged from 1.22 microg/mL to 39.06 microg/mL for Mycobacterium smegmatis and all studied Mycobacterium tuberculosis strains for the crude extract from D. crassiflora, diospyrone and crassiflorone. Results of the killing rate experiment revealed that a total inhibition effect on M. tuberculosis H37Rv strain was observed at Day 18 for D. crassiflora and Day 21 for the crude extract from D. canaliculata and diospyrone at 4x MIC as determined by MABA. Results of the antigonorrhoeal assay indicated that diospyrone was able to prevent the growth of all studied strains of Neisseria gonorrhoeae. The overall results of this work provide evidence that the studied plant extracts (diospyrone, crassiflorone and plumbagin) might be potential sources of new antimicrobial drugs against tuberculosis and gonorrhoea.

Topics: Anti-Bacterial Agents; Antitubercular Agents; Diospyros; Microbial Sensitivity Tests; Mycobacterium smegmatis; Mycobacterium tuberculosis; Naphthoquinones; Neisseria gonorrhoeae; Plant Extracts

Plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica has been shown to exert anticancer and anti-proliferative activities in cells in culture as well as animal tumor models. In our previous paper, we have reported the cytotoxic action of plumbagin in plasmid pBR322 DNA as well as human peripheral blood lymphocytes through a redox mechanism involving copper. Copper has been shown to be capable of mediating the action of several plant-derived compounds through production of reactive oxygen species (ROS). The objective of the present study was to determine whether plumbagin induces apoptosis in human cancer cells through the same mechanism which we proposed earlier. Using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, 3-(4,5-B-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for cell growth inhibition, histone/DNA ELISA, homogeneous caspase-3/7 assay for apoptosis as well as alkaline comet assay for DNA single-strand breaks detection in this report, we confirm that plumbagin causes effective cell growth inhibition, induces apoptosis and generates single-strand breaks in cancer cells. Incubation of cancer cells with scavengers of ROS and neocuproine inhibited the cytotoxic action of plumbagin proving that generation of ROS and Cu(I) are the critical mediators in plumbagin-induced cell growth inhibition. This study is the first to investigate the copper-mediated anticancer mechanism of plumbagin in human cancer cells and these properties of plumbagin could be further explored for the development of anticancer agents with higher therapeutic indices, especially for skin cancer.

Topics: Cell Death; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Shape; Comet Assay; Copper; Cytoprotection; Free Radical Scavengers; Humans; Naphthoquinones; Neoplasms; Oxidation-Reduction; Phenanthrolines; Skin Neoplasms

A series of plumbagin derivatives (4a-4k) containing an amino acid moiety were synthesized under mild esterification conditions in excellent yields (35%-80%) and screened for their antifeedant activities in tobacco caterpillar (Spodoptera litura) and castor semilooper (Achaea janata) using a no-choice laboratory bioassay. The parent compound plumbagin lacked significant activity, but the analogues were effective in reducing feeding by two insect species. The introduction of an N-acetyl-l-amino acid side chain to the Michael adduct of plumbagin at the third position of the quinone moiety significantly increased antifeedant activity. Several of the analogues were also toxic or caused developmental abnormalities following topical administration.

Topics: Animals; Benzoquinones; Eating; Insecticides; Larva; Moths; Naphthoquinones; Spodoptera; Structure-Activity Relationship; Superoxides

Female adult bovine filarial worms Setaria digitata were extracted with phosphate-buffered saline (pH 7.4) and glutathione S-transferase (GST) activity and protein content were determined. The protein content, GST enzyme activity, and specific activity were 10.61 +/- 3.41 mg ml(-1), 0.09 +/- 0.019 micromol min(-1) ml(-1), and 0.009 +/- 0.002 micromol min(-1) mg(-1) protein, respectively. The GST inhibition studies were performed with and without the inhibitors resulted from earlier molecular docking studies viz., ethacrynic acid, plumbagin, and curcumin for which the IC(50) values were 19.42, 51.41, and 114.86 microM, respectively. The in vitro macrofilaricidal activity of these molecules was studied by worm motility and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay at 24- and 48-h incubation. Plumbagin and ethacrynic acid showed 100% inhibition in worm motility at lower concentrations of 3.19 and 6.6 microM, respectively, at 48-h incubation while curcumin was effective at 54.29 microM. In MTT reduction assay, the ED(50) values (50% inhibition in formazan formation) for plumbagin, ethacrynic acid, and curcumin at 48-h incubation were 1.20, 2.48, and 19.86 microM, respectively. MTT reduction assay showed that plumbagin was the most effective in killing the adult S. digitata worms followed by ethacrynic acid and curcumin. In conclusion, all the three molecules selected by molecular modeling and docking studies inhibited the GST enzyme isolated from S. digitata and exhibited macrofilaricidal activity in vitro.

Topics: Animals; Cattle; Cattle Diseases; Curcumin; Ethacrynic Acid; Female; Filariasis; Filaricides; Filarioidea; Glutathione Transferase; Helminth Proteins; Inhibitory Concentration 50; Locomotion; Naphthoquinones; Survival Analysis

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Topics: Acetylation; Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Enzyme Inhibitors; Humans; Male; Mice; Mutagenesis, Site-Directed; Naphthoquinones; p300-CBP Transcription Factors; Plant Roots; Plumbaginaceae; Protein Structure, Tertiary; Tumor Suppressor Protein p53

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

Superoxide and its products, especially hydroxyl radical, were recently proposed to be instrumental in cell death following treatment with a wide range of antimicrobials. Surprisingly, bleomycin lethality to Escherichia coli was ameliorated by a genetic deficiency of superoxide dismutase or by furnishing the superoxide generator plumbagin. Rescue by plumbagin was similar in strains containing or lacking recA or with inactive, inducible, or constitutive soxRS regulons. Thus, superoxide interferes with bleomycin cytotoxicity in ways not readily explained by genetic pathways expected to protect from oxidative damage.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Bleomycin; Escherichia coli; Escherichia coli Proteins; Mutant Proteins; Naphthoquinones; Rec A Recombinases; Superoxide Dismutase; Superoxides; Trans-Activators; Transcription Factors; Transduction, Genetic; Transformation, Bacterial

Inducible defenses against oxidative stress are coordinated by redox-sensitive transcription factors that transduce oxidative damage into differential gene expression. The opportunistic human pathogen Pseudomonas aeruginosa has evolved under physiological and host-derived sources of oxidative stress. Previous work showed that the pqrABC and pqrR genes of P. aeruginosa, all lacking known functions, were induced by treatment of three different isolates of P. aeruginosa with paraquat (PQ), a superoxide-producing agent. Insertional mutation of the pqrABCR genes resulted in hypersensitive phenotypes to a variety of oxidants, although the hypersensitivity to PQ was marginal. Mutation of pqrR and complementation assays showed that PqrR regulated the pqrABC genes in response to PQ. PqrR, a member of the MarR family of transcriptional regulators, contains a C-terminal region with four conserved cysteines, which suggested redox-regulated transcriptional activity. Purified PqrR bound to two discrete sites at the pqrA and pqrR regulatory regions. The in vitro DNA binding activity of PqrR was decreased by exposure to air and reconstituted by treatment with dl-dithiothreitol. Elemental analysis and preliminary electron paramagnetic resonance experiments showed that PqrR contains iron. Interestingly, site-directed mutagenesis of C-terminal cysteines demonstrated that the four conserved cysteine residues are essential for in vivo redox sensing by PqrR.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Diamide; Gene Expression Regulation, Bacterial; Iron; Molecular Sequence Data; Mutation; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Paraquat; Pseudomonas aeruginosa; Signal Transduction; Silencer Elements, Transcriptional; Transcription, Genetic

The anticancer traditional Chinese medicine (TCM), plumbagin (PLN), was isolated from Plumbago Zeylanica. Reaction of plumbagin with Cu(II) salt, afforded [Cu(PLN)(2)].2H(2)O (1). With 2,2'-bipyridine (bipy) as a co-ligand, PLN reacts with Cu(II) to give [Cu(PLN)(bipy)(H(2)O)](2)(NO(3))(2).4H(2)O (2). 1 and 2 were characterized by elemental analysis, IR, ESI-MS spectra. Their crystal structures were determined by single crystal X-ray diffraction methods. The in vitro cytotoxicity of PLN, 1 and 2 against seven human tumour cell lines was assayed. The metal-based compounds exhibit enhanced cytotoxicity vs. that of free PLN, suggesting that these compounds display synergy in the combination of metal ions with PLN. The binding properties of PLN, 1 and 2 to DNA were investigated through UV-vis, fluorescence, CD spectra, and gel mobility shift assay, which indicated that 1 and 2 were non-covalent binding and mainly intercalated the neighboring base pairs of DNA. PLN, 1 and 2 exhibit inhibition activity to topoisomerase I (TOPO I), but 1 and 2 were more effective than PLN.

Topics: 2,2'-Dipyridyl; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Coordination Complexes; Copper; Crystallography, X-Ray; DNA; DNA Topoisomerases, Type I; Drug Screening Assays, Antitumor; Humans; Medicine, Chinese Traditional; Molecular Conformation; Naphthoquinones; Plant Roots; Plumbaginaceae; Topoisomerase I Inhibitors

Pancreatic cancer is one of the most resistant malignancies. Several studies have indicated that plumbagin isolated from Plumbago zeylanica possesses anticancer activity. However, its antitumor effects against pancreatic cancer have not been explored.. We investigated the effect of plumbagin on the growth of human pancreatic carcinoma cells and its possible underlying mechanisms.. Plumbagin inhibited the growth of Panc-1 and Bxpc-3 cells in a dose-dependent and time-dependent manner. Liu's staining and transmission electron microscopy demonstrated morphological changes resembling apoptosis in Panc-1 cells treated with plumbagin. The degree of apoptosis was assessed by measuring the proportions of sub-G(1), annexin V+/propidium iodide-, and terminal- deoxynucleotidyl-transferase-mediated-nick-end labeling (TUNEL)+ cells, and a significant increment in apoptotic cells was observed. Exposure to plumbagin caused the upregulation of Bax, a rapid decline in mitochondrial transmembrane potential, apoptosis-inducing factor overexpression in cytosol, and the cleavage of procaspase-9 and poly ADP-ribose polymerase. Activation of caspase-3, but not caspase-8, was evidenced by fluorometric substrate assay. Pretreatment with caspase inhibitors did not block plumbagin-induced apoptosis. Alternatively, it is possible that plumbagin downregulated phosphoinositide 3-kinase activity through a negative feedback mechanism. In an orthotopic pancreatic tumor model, plumbagin markedly inhibited the growth of Panc-1 xenografts without any significant effect on leukocyte counts or body weight.. Plumbagin may induce apoptosis in human pancreatic cancer cells primarily through the mitochondria-related pathway followed by both caspase-dependent and caspase-independent cascades. It indicates that plumbagin can be potentially developed as a novel therapeutic agent against pancreatic cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma; Caspases; Cell Line, Tumor; Cell Proliferation; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Naphthoquinones; Pancreatic Neoplasms; Plumbaginaceae; Xenograft Model Antitumor Assays

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Goats; Humans; Kinetics; Microscopy, Confocal; Microscopy, Electron, Transmission; Microtubules; Molecular Structure; Naphthoquinones; Protein Binding; Spectrometry, Fluorescence; Tubulin

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. Hormone-refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. We present here that plumbagin (PL), a quinoid constituent isolated from the root of the medicinal plant Plumbago zeylanica L., may be a potential novel agent in the control of hormone-refractory PCa. Specific observations are the findings that PL inhibited PCa cell invasion and selectively induced apoptosis in PCa cells but not in immortalized nontumorigenic prostate epithelial RWPE-1 cells. In addition, i.p. administration of PL (2 mg/kg body weight), beginning 3 days after ectopic implantation of hormone-refractory DU145 PCa cells, delayed tumor growth by 3 weeks and reduced both tumor weight and volume by 90%. Discontinuation of PL treatment in PL-treated mice for as long as 4 weeks did not result in progression of tumor growth. PL, at concentrations as low as 5 micromol/L, inhibited in both cultured PCa cells and DU145 xenografts (a) the expression of protein kinase Cepsilon (PKCepsilon), phosphatidylinositol 3-kinase, phosphorylated AKT, phosphorylated Janus-activated kinase-2, and phosphorylated signal transducer and activator of transcription 3 (Stat3); (b) the DNA-binding activity of transcription factors activator protein-1, nuclear factor-kappaB, and Stat3; and (c) Bcl-xL, cdc25A, and cyclooxygenase-2 expression. The results indicate for the first time, using both in vitro and in vivo preclinical models, that PL inhibits the growth and invasion of PCa. PL inhibits multiple molecular targets including PKCepsilon, a predictive biomarker of PCa aggressiveness. PL may be a novel agent for therapy of hormone-refractory PCa.

Topics: Animals; Apoptosis; Base Sequence; Cell Division; DNA Primers; Electrophoretic Mobility Shift Assay; Humans; Immunohistochemistry; Male; Mice; Mice, Nude; Naphthoquinones; Neoplasm Invasiveness; Plants, Medicinal; Prostatic Neoplasms

Breast cancer remains the major cause of cancer-related deaths in women world-wide. The heterogeneity of breast cancer has further complicated the progress of target-based therapies. Triple negative breast cancers, lacking estrogen receptor, progesterone receptor and the Her-2/neu (ErbB2), represent a highly aggressive breast cancer subtype, that are difficult to treat. Pleiotropic agents, such as those found in nature, can target receptor-positive as well as receptor-negative cancer cells, suggesting that such agents could have significant impact in breast cancer prevention and/or therapy. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is one such agent which has anti-tumor activity against several cancers. However, its mechanism of action against breast cancer is not clearly understood. We hypothesized that plumbagin may act as an effective agent against breast cancer especially triple negative breast cancer. We tested our hypothesis using ER-positive MCF-7 and ER-negative MDA-MB-231 (triple negative) breast cancer cells, and we found that plumbagin significantly inhibits the growth of breast cancer cells with no effect on normal breast epithelial cells. We also found that plumbagin induces apoptosis with concomitant inactivation of Bcl-2 and the DNA binding activity of NF-kappaB. Bcl-2 over-expression resulted in attenuation of plumbagin-induced effects, suggesting that the inhibition of cell growth and induction of apoptosis by plumbagin is in part due to inactivation of NF-kappaB/Bcl-2 pathway. To our knowledge, this is the first report, showing mechanistic and cancer cell specific apoptosis-inducing effects of plumbagin in breast cancer cells, suggesting the potential role of plumbagin in the prevention and/or treatment of breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Female; Humans; Naphthoquinones; NF-kappa B; Proto-Oncogene Proteins c-bcl-2

To establish the qualitative and quantitative methods of Caratostigma willmottianum.. The roots of Radix Ceratostigmae were identified by TLC with the authentic medicinal material and plumbagin as the reference. The contents of plumbagin in Radix Ceratostigmae were determined by HPLC.. The TLC method was simple and specific. A Diamonsil C18 column was used. The mobile phase was methanol-water (75:25). Plumbagin in the sawple extract was separated well. The linear range of plumbagin was 33.6-313.6 ng. The average recovery of plumbagin was 100.3% and RSD was 1.9%.. The methods can be used for identification and determination of plumbagin in C. willmottianum.

Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Drugs, Chinese Herbal; Naphthoquinones; Plumbaginaceae

Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation.. Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Delta and gtt2Delta) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin.. These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification.

Topics: Antineoplastic Agents, Phytogenic; Glutathione; Glutathione Transferase; Lipid Peroxidation; Microbial Sensitivity Tests; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Saccharomyces cerevisiae; Vitamin K 3; Vitamins

Radiotherapy is the primary line of cancer treatment for cervical cancer and is known to induce cell death in tumors. Radiotherapy is however limited by the total dose that can be given without damaging normal tissue. Plumbagin, a naturally occurring naphthaquinone, has been reported to have free radical producing properties. Hence we hypothesized that plumbagin could also have properties that could modify effects of radiation on cervical cancer cells. Radiation in combination with plumbagin may thus have treatment augmenting effects. Results from our studies have shown that a lower dose of radiation in combination with plumbagin could induce apoptosis more effectively compared to a higher dose of radiation alone. Plumbagin in combination with 2 Gy of radiation was very effective in inducing apoptosis, when compared to a higher radiation dose of 10 Gy alone. This combination also showed a fivefold increase in the activation of caspase 3 in C33A cells. Activation of effector caspases confirms that the induction of apoptosis by irradiation and plumbagin involves caspase-dependent pathways. Expression of apoptotic regulatory molecules Bcl-2, Bax and Survivin was also modulated by plumbagin in combination with radiation. In summary, this study shows that a combination of plumbagin and radiation augmented cell growth inhibition compared to higher radiation dose alone, thus indicating that plumbagin may be a potential radiosensitizer acting through the induction of apoptosis.

Topics: Apoptosis; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Female; Gamma Rays; HeLa Cells; Humans; Membrane Potentials; Mitochondrial Membranes; Naphthoquinones; Radiation-Sensitizing Agents; Uterine Cervical Neoplasms

This study is the first to investigate the anticancer effect of plumbagin in human melanoma A375.S2 cells. Plumbagin exhibited effective cell growth inhibition by inducing cancer cells to undergo S-G2/M phase arrest and apoptosis. Further investigation revealed that plumbagin's inhibition of cell growth was also evident in a nude mice model. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. Plumbagin triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activation. We also found the generation of ROS is a critical mediator in plumbagin-induced cell growth inhibition. Plumbagin increased the activation of apoptosis signal-regulating kinase 1, JNK and extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38. In addition, antioxidants vitamin C and catalase significantly decreased plumbagin-mediated c-Jun N-terminal kinase (JNK) activation and apoptosis. Moreover, blocking ERK and JNK by specific inhibitors suppressed plumbagin-triggered mitochondrial apoptotic pathway. Taken together, these results imply a critical role for ROS and JNK in the plumbagin's anticancer activity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Activation; Glutathione; Humans; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase Kinase 5; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; Neoplastic Stem Cells; Phosphorylation; Reactive Oxygen Species; Signal Transduction; Time Factors; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

The roles of SitABCD, MntH, and FeoB metal transporters in the virulence of avian pathogenic Escherichia coli (APEC) O78 strain chi7122 were assessed using isogenic mutants in chicken infection models. In a single-strain infection model, compared to chi7122, the Deltasit strain demonstrated reduced colonization of the lungs, liver, and spleen. Complementation of the Deltasit strain restored virulence. In a coinfection model, compared to the virulent APEC strain, the Deltasit strain demonstrated mean 50-fold, 126-fold, and 25-fold decreases in colonization of the lungs, liver, and spleen, respectively. A DeltamntH Deltasit strain was further attenuated, demonstrating reduced persistence in blood and mean 1,400-fold, 954-fold, and 83-fold reduced colonization in the lungs, liver, and spleen, respectively. In coinfections, the DeltafeoB Deltasit strain demonstrated reduced persistence in blood but increased colonization of the liver. The DeltamntH, DeltafeoB, and DeltafeoB DeltamntH strains were as virulent as the wild type in either of the infection models. Strains were also tested for sensitivity to oxidative stress-generating agents. The DeltamntH Deltasit strain was the most sensitive strain and was significantly more sensitive than the other strains to hydrogen peroxide, plumbagin, and paraquat. sit sequences were highly associated with APEC and human extraintestinal pathogenic E. coli compared to commensal isolates and diarrheagenic E. coli. Comparative genomic analyses also demonstrated that sit sequences are carried on conjugative plasmids or associated with phage elements and were likely acquired by distinct genetic events among pathogenic E. coli and Shigella sp. strains. Overall, the results demonstrate that SitABCD contributes to virulence and, together with MntH, to increased resistance to oxidative stress.

Topics: Animals; Anti-Bacterial Agents; Bacteriophages; Blood; Carrier Proteins; Cation Transport Proteins; Chickens; Colony Count, Microbial; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Gene Deletion; Genetic Complementation Test; Hydrogen Peroxide; Metals; Naphthoquinones; Paraquat; Plasmids; Poultry Diseases; Virulence; Virulence Factors

Plumbagin (PL; 5-hydroxy-2-methyl-1,4-naphthoquinone) is a natural compound mainly isolated from Plumbago zeylanica. This plant is distributed in Southeast Asia, and well known as Ayurvedic medicine in India for its medicinal properties. PL has been shown to have various pharmacological activities. We have successfully prepared monoclonal antibodies against PL, and developed an enzyme-linked immunosorbent assay (ELISA) system for determination of PL. 3-(5-Hydroxy-2-methyl-1,4-naphthoquinone-3-yl) propanoic acid was synthesized and purified to prepare PL-bovine serum albumin conjugate (PL-BSA), which was used as an immunogen. PL-BSA conjugate was administered into BALB/c male mice for production of monoclonal antibodies against PL. The monoclonal antibody against PL which is secreted from established hybridoma cell line 3A3 (MAb 3A3) has been proven to have highly-specific to PL resulting from cross-reactivities test. The range for calibration of PL by ELISA was 0.2-25 microg mL(-1). Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of PL in plant.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Carrier Proteins; Cattle; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Haptens; Mice; Naphthoquinones; Plumbaginaceae

Cellular response to chemotherapeutic drugs in the absence of BRCA1 either completely or partially had drawn less attention. The present study evaluated whether there is a differential inhibition of cell growth by selected compounds with respect to BRCA1 status in estrogen receptor (ER)-positive ovarian cancer cells.. The BG1 ovarian cancer cells used in the experiments were antisensely blocked with BRCA1 gene. Growth inhibition and apoptotic induction were analyzed to evaluate the cytotoxic effects. Small interfering RNA (SiRNA) transfection, western blot analysis, RT-PCR analysis and molecular modeling were carried out to analyze the estrogen-dependent action of plumbagin.. Although we found that all the compounds studied induce apoptosis, the induction was in the order of plumbagin > doxorubicin > tamoxifen > cisplatin. Plumbagin can bind to the active site of ER-alpha. Plumbagin, however, induced ER-alpha 46 kDa truncated isoform, which was found abundantly preempted in the cytoplasm compared with a 66-kDa full-length isoform. The truncated isoform is known to inhibit classical ER-alpha signaling pathways. SiRNA-transfected cells for ER-alpha exhibited lower cytotoxicity upon plumbagin treatment than the control-transfected cells.. Taken together, this study indicates that plumbagin has chemotherapeutic potential in BRCA1-mutated/defective ER-positive cancers.

Topics: Actins; Adenocarcinoma; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Cisplatin; Doxorubicin; Estrogen Receptor alpha; Female; Genes, BRCA1; Humans; Lethal Dose 50; Naphthoquinones; Ovarian Neoplasms; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Tamoxifen; Transfection; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

To investigate the mechanism of human prostate cancer cell growth inhibition by plumbagin, a constituent of the widely used medicinal herb Plumbago zeylanica L.. Cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Cell cycle distribution and generation of reactive oxygen species (ROS) were determined by flow cytometry. The effect of plumbagin treatment on cellular redox status was determined by analysis of intracellular glutathione (GSH) levels and expression of genes involved in ROS metabolism.. Plumbagin treatment decreased viability of human prostate cancer cells (PC-3, LNCaP, and C4-2) irrespective of their androgen responsiveness or p53 status. Plumbagin-mediated decrease in cell viability correlated with apoptosis induction, which was accompanied by ROS generation and depletion of intracellular GSH levels. Pretreatment of cells with the antioxidant N-acetylcysteine inhibited plumbagin-mediated ROS generation and apoptosis. Plumbagin treatment also resulted in altered expression of genes responsible for ROS metabolism, including superoxide dismutase 2 (Mn-SOD).. The present study points towards an important role of ROS in plumbagin-induced apoptosis in human prostate cancer cells.

Topics: Acetylcysteine; Anticarcinogenic Agents; Antioxidants; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Male; Naphthoquinones; Oxidation-Reduction; Peroxidases; Prostatic Neoplasms; Reactive Oxygen Species; Superoxide Dismutase

The pathophysiology of rheumatoid arthritis (RA) includes inflammation, synoviocyte proliferation, angiogenesis, and matrix metalloproteinase-driven degradation processes. The objective of this study was to investigate a variety of structurally unrelated anticancer topoisomerase inhibiting agents as inhibitors of aspects of these disease processes involved in RA.. The topoisomerase I inhibitors camptothecin and beta-laperchone and the topoisomerase II inhibitors, etoposide, doxorubicin, plumbagin and menadione were used in this study. Crystal induced neutrophil activation was measured by luminol dependent chemiluminescence. Synoviocyte proliferation was measured by an MTT assay using HIG 82 rabbit synoviocytes in cell culture. Angiogenesis was measured using the chorioallantoic membrane of the chick embryo. Chondrocyte (culture primary cells) expression of the matrix metalloproteinases collagenase and stromelysin was measured by Northern Blot analysis.. All agents inhibited synoviocyte proliferation to some degree. Camptothecin had no effect on neutrophil activation but inhibited all other processes at low (nanomolar) concentrations. Plumbagin and menadione inhibited neutrophil activation, collagenases expression and angiogenesis. The other agents had little effect on neutrophil activation (except beta-laperchone) but inhibited angiogenesis and collagenase expression to a lesser degree than camptothecin.. These studies support the explorative use of topoisomerase I (particularly camptothecin) and II inhibitors as potential agents for use against RA.

Topics: Animals; Antirheumatic Agents; Arthritis, Rheumatoid; Camptothecin; Cattle; Cell Proliferation; Cells, Cultured; Chick Embryo; Chondrocytes; Doxorubicin; Enzyme Inhibitors; Etoposide; Interleukin-1; Naphthoquinones; Neutrophils; Rabbits; Synovial Membrane; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors; Vitamin K 3

Acaricidal effects of materials derived from Diospyros kaki roots against Dermatophagoides farinae and D. pteronyssinus were assessed using impregnated fabric disk bioassay and compared with that of the commercial benzyl benzoate. The observed responses varied according to dosage and mite species. The LD50 values of the chloroform extract of Diospyros kaki roots were 1.66 and 0.96 microg/cm2 against D. farinae and D. pteronyssinus. The chloroform extract of Diospyros kaki roots was approximately 15.2 more toxic than benzyl benzoate against D. farinae, and 7.6 times more toxic against D. pteronyssinus. Purification of the biologically active constituent from D. kaki roots was done by using silica gel chromatography and high-performance liquid chromatography. The structure of the acaricidal component was analyzed by GCMS, 1H-NMR, 13C-NMR, 1H-13C COSY-NMR, and DEPTNMR spectra, and identified as plumbagin. The acaricidal activity of plumbagin and its derivatives (naphthazarin, dichlon, 2,3-dibromo-1,4-naphthoquinone, and 2-bromo-1,4- naphthoquinone) was examined. On the basis of LD50 values, the most toxic compound against D. farinae was naphthazarin (0.011 microg/cm2) followed by plumbagin (0.019 microg/cm2), 2- bromo-1,4-naphthoquinone (0.079 microg/cm2), dichlon (0.422 microg/ cm2), and benzyl benzoate (9.14 microg/cm2). Additionally, the skin color of the dust mites was changed from colorless-transparent to dark brown-black by the treatment of plumbagin. Similar results have been exhibited in its derivatives (naphthazarin, dichlon, and 2-bromo-1,4-naphthoquinone). In contrast, little or no discoloration was observed for benzyl benzoate. From this point of view, plumbagin and its derivatives can be very useful for the potential control agents, lead compounds, and indicator of house dust mites.

Topics: Animals; Benzoates; Diospyros; Gas Chromatography-Mass Spectrometry; Insecticides; Lethal Dose 50; Naphthoquinones; Plant Extracts; Plant Roots; Pyroglyphidae

Streptomyces reticuli is a soil-growing Gram-positive bacteria that has been shown to secrete a novel haem-binding protein known as HbpS. Sequence analysis reveals that homologues of HbpS are found in a wide variety of bacteria, including different Actinobacteria and the Gram-negative Vibrio cholera and Klebsiella pneumoniae. The in vivo production of HbpS is greatly increased when S. reticuli is cultured in the presence of the natural antibiotic haemin (Fe3+ oxidized form of haem). Mutational analysis demonstrated that HbpS significantly increases the resistance of S. reticuli to toxic concentrations of haemin. Previous data show that the presence of the newly identified two-component sensor system SenS-SenR also considerably enhances the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting a role in the sensing of redox changes. Specific interaction between HbpS and SenS-SenR, which regulates the expression of the catalase-peroxidase CpeB, as well as HbpS, has been demonstrated in vitro. HbpS has been recombinantly overexpressed, purified and crystallized in space group P2(1)3, with a cell edge of 152.5 A. Diffraction data were recorded to a maximal resolution of 2.25 A and phases were obtained using the SAD method from crystals briefly soaked in high concentrations of sodium bromide.

Topics: Bacterial Proteins; Carrier Proteins; Cloning, Molecular; Crystallization; Electrophoresis, Agar Gel; Escherichia coli; Heme-Binding Proteins; Hemeproteins; Hemin; Naphthoquinones; Oxidation-Reduction; Peroxidases; Streptomyces; X-Ray Diffraction

Plumbago indica L. contains naphthoquinones that are derived from six acetate units. To characterize the enzyme catalyzing the first step in the biosynthesis of these metabolites, a cDNA encoding a type III polyketide synthase (PKS) was isolated from roots of P. indica. The translated polypeptide shared 47-60% identical residues with PKSs from other plant species. Recombinant P. indica PKS expressed in Escherichia coli accepted acetyl-CoA as starter and carried out five decarboxylative condensations with malonyl coenzyme A (-CoA). The resulting hexaketide was not folded into a naphthalene derivative. Instead, an alpha-pyrone, 6-(2',4'-dihydroxy-6'-methylphenyl)-4-hydroxy-2-pyrone, was produced. In addition, formation of alpha-pyrones with linear keto side chains derived from three to six acetate units was observed. As phenylpyrones could not be detected in P. indica roots, we propose that the novel PKS is involved in the biosynthesis of naphthoquinones, and additional cofactors are probably required for the biosynthesis of these secondary metabolites in vivo.

Topics: Amino Acid Sequence; Carbon; Catalysis; DNA, Complementary; Gas Chromatography-Mass Spectrometry; Malonyl Coenzyme A; Models, Chemical; Molecular Sequence Data; Naphthoquinones; Phylogeny; Plumbaginaceae; Polyketide Synthases; Pyrones; Quinones; Sequence Homology, Amino Acid

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

In this study the methanol/dichloromethane (1:1) extract and plumbagin isolated from extract of stem barks of Diospyros crassiflora were tested for their antifungal activity against 12 strains of yeast pathogens and filamentous fungi: Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Cryptococcus neoformans, Aspergillus niger, Aspergillus flavus, Alternaria sp., Cladosporium sp., Geotrichum candidum, Fusarium sp. and Penicillium sp. The growth of all fungi strains tested was inhibited by the extract and plumbagin. The diameter of inhibition zones varied from 12 to 18 mm and from 21 to 35 mm for the extract and plumbagin, respectively. The MIC values ranged from 12.5 to 25 mg/mL for the extract and 0.78-3.12 microg/mL for plumbagin. It is therefore suggested that extracts from the stem bark of Diospyros crassiflora could be used traditionally in the treatment of fungal infections. Compared with ketoconazole used as a standard antifungal, plumbagin could be considered as a promising antifungal agent.

Topics: Antifungal Agents; Diospyros; Humans; Microbial Sensitivity Tests; Mitosporic Fungi; Naphthoquinones; Phytotherapy; Plant Extracts; Plant Stems

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topo II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.

Topics: Apoptosis; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Drosera; Enzyme Inhibitors; Flow Cytometry; Gas Chromatography-Mass Spectrometry; HL-60 Cells; Humans; Naphthoquinones; Reactive Oxygen Species; Topoisomerase II Inhibitors

Mycobacteria can tolerate relatively high concentrations of triphenylmethane dyes such as malachite green and methyl violet. To identify mycobacterial genes involved in the decolorization of malachite green, a transposon mutant library of Mycobacterium smegmatis mc2 155 was screened for mutants unable to decolorize this dye. One of the genes identified was MSMEG_5126, an orthologue of Mycobacterium bovis fbiC encoding a 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) synthase, which is essential for the biosynthesis of the electron carrier coenzyme F420. The other gene identified was MSMEG_2392, encoding an alanine-rich protein with a DUF121 domain. The minimum inhibitory concentrations (MICs) for malachite green and methyl violet of the six fbiC mutants and two MSMEG_2392 mutants were one-third and one-fifth, respectively, of the MIC of the parent strain M. smegmatis mc2 155. Representative fbiC and MSMEG_2392 mutant strains were also sensitive to oxidative stress caused by the redox-cycling agents plumbagin and menadione, and the sensitivity was reversed in the complemented strains. HPLC analysis of representative fbiC and MSMEG_2392 strains revealed that, while the fbiC mutant lacked both coenzyme F420 and FO, the MSMEG_2392 mutant contained FO but not coenzyme F420. These results indicate that MSMEG_2392 is involved in the biosynthesis of coenzyme F420.

Topics: Anti-Bacterial Agents; Coloring Agents; DNA Transposable Elements; Gene Deletion; Genetic Complementation Test; Gentian Violet; Microbial Sensitivity Tests; Mutagenesis, Insertional; Mycobacterium smegmatis; Naphthoquinones; Riboflavin; Riboflavin Synthase; Rosaniline Dyes; Vitamin K 3

To investigate the chemical constituents of the aerial parts of Plumbago zeylanica Linn.. The constituents of the EtOAc-soluble portion in the 95% ethanol extract were isolated and purified by means of chromatography. Compounds were identified by their physical characteristics and spectral features.. Nine compounds were isolated as plumbagin (I), isoshinanolone (II), plumbagic acid (III), beta-sitosterol (IV), 4-hydroxybenzaldehyde (V), trans-cinnamic acid (VI), vanillic acid (VII), 2, 5-dimethyl-7-hydroxychromone (VIII), indole-3-carboxaldehyde (IX).. Compounds V, VII, VIII and IX were isolated for the first time from Plumbago Linn.

Topics: Benzaldehydes; Cinnamates; Indoles; Magnetic Resonance Spectroscopy; Molecular Structure; Naphthoquinones; Plant Components, Aerial; Plants, Medicinal; Plumbaginaceae; Sitosterols; Tetrahydronaphthalenes; Vanillic Acid

Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2, which are essential for blood clotting. The naturally occurring structural analogue of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We here report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). Vitamin K3 and plumbagin inhibited the binding of [(125)I]iodoarylazidoprazosin, a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC(50) values of 7.3 and 22.6 micromol/L, respectively, but had no effect on the binding of the photoaffinity analogue to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of the ABCG2 transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared with the control cells, suggesting that they are substrates of this transporter. Collectively, these data show for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Cholecalciferol; Drug Resistance, Multiple; Humans; Hydrolysis; Mitoxantrone; Naphthoquinones; Neoplasm Proteins; Photoaffinity Labels

Plumbagin is found in many medicinal plants and has been reported to have antimicrobial activities. We examined the molecular responses of Escherichia coli to plumbagin by using a proteomic approach to search for bacterial genes up-regulated by the drug. The protein profile obtained was compared with that of E. coli without the plumbagin treatment. Subsequent analyses of the induced proteins by mass spectroscopy identified several up-regulated genes, including ygfZ, whose function has not been defined. Analyses of the 5'-flanking sequences indicate that most of these genes contain a marbox-like stretch, and several of them are categorized as members of the mar/sox regulon. Representatives of these genes were cloned into plasmids, and the marbox-like sequences were modified by site-directed mutagenesis. It was proven that mutations in these regions substantially repressed the level of proteins encoded by the downstream genes. Furthermore, plumbagin's early effect was demonstrated to robustly induce SoxS rather than MarA, an observation distinctly different from that seen with sodium salicylate.

Topics: Amino Acid Sequence; Consensus Sequence; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Regulator; Molecular Sequence Data; Mutation; Naphthoquinones; Peroxidases; Peroxiredoxins; Trans-Activators; Up-Regulation

Plumbagin, derived from the medicinal plant Plumbago zeylanica, modulates cellular proliferation, carcinogenesis, and radioresistance, all known to be regulated by the activation of the transcription factor NF-kappaB, suggesting plumbagin might affect the NF-kappaB activation pathway. We found that plumbagin inhibited NF-kappaB activation induced by TNF, and other carcinogens and inflammatory stimuli (e.g. phorbol 12-myristate 13-acetate, H2O2, cigarette smoke condensate, interleukin-1beta, lipopolysaccharide, and okadaic acid). Plumbagin also suppressed the constitutive NF-kappaB activation in certain tumor cells. The suppression of NF-kappaB activation correlated with sequential inhibition of the tumor necrosis factor (TNF)-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRAF2, NIK, IKK-beta, and the p65 subunit of NF-kappaB. Plumbagin also suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by dithiothreitol both in vitro and in vivo. However, plumbagin did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine 38 mutated to serine. Plumbagin down-regulated the expression of NF-kappaB-regulated anti-apoptotic (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin), proliferative (cyclin D1 and COX-2), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by TNF and paclitaxel and inhibited cell invasion. Overall, our results indicate that plumbagin is a potent inhibitor of the NF-kappaB activation pathway that leads to suppression of NF-kappaB-regulated gene products. This may explain its cell growth modulatory, anticarcinogenic, and radiosensitizing effects previously described.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cytokines; Dithiothreitol; Enzyme Activation; Humans; I-kappa B Proteins; Inflammation; Models, Chemical; Naphthoquinones; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; U937 Cells

This study first investigates the anticancer effect of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) in human nonsmall cell lung cancer cells, A549. Plumbagin has exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased levels of p21 and reduced amounts of cyclinB1, Cdc2, and Cdc25C. Plumbagin treatment also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. Blockade of p53 activity by dominant-negative p53 transfection partially decreased plumbagin-induced apoptosis and G2/M arrest, suggesting it might be operated by p53-dependent and independent pathway. Plumbagin treatment triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and caspase-9 activation. We also found that c-Jun NH2-terminal kinase (JNK) is a critical mediator in plumbagin-induced cell growth inhibition. Activation of JNK by plumbagin phosphorylated p53 at serine 15, resulting in increased stability of p53 by decreasing p53 and MDM2 interaction. SP600125 (anthra [1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone), a specific inhibitor of JNK, significantly decreased apoptosis by inhibiting the phosphorylation of p53 (serine 15) and subsequently increased the interaction of p53 and MDM2. SP6000125 also inhibited the phosphorylation of Bcl-2 (Ser70) induced by plumbagin. Further investigation revealed that plumbagin's inhibition of cell growth effect was also evident in a nude mice model. Taken together, these results suggest a critical role for JNK and p53 in plumbagin-induced G2/M arrest and apoptosis of human nonsmall cell lung cancer cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 9; Caspases; Cell Cycle; Cell Line; Cell Proliferation; Humans; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mice; Mice, Nude; Mitochondria; Naphthoquinones; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Serine; Signal Transduction; Transfection; Tumor Stem Cell Assay; Tumor Suppressor Protein p53

According to previous clinical experiences of the authors, plumbago zeylanica was effective against acute promyelocytic leukemia (APL). However, its effectiveness has never been proven experimentally or unequivocally clinically. This study was aimed to investigate the effects of plumbagin on the proliferation, cell cycle and apoptosis of APL cell line NB4 Cells. Cell inhibitory rates were detected by MTT colorimetric assay; morphologic changes were observed under light microscope and transmission electron microscope; apoptosis-inducing effects were determined by DNA gel electrophoresis, annexin V/PI double-stained and PI single-stained flow cytometry. The results demonstrated that 2-15 micromol/L plumbagin inhibited the proliferation of NB4 cells in a dose-dependent manner. The morphologic changes of cell apoptosis, such as chromsome condensation and apoptotic body formation, were observed by light microscope and transmission electron microscope. Cell cycle analysis showed that NB4 cells were blocked in G2/M phase of cell cycle. And plumbagin induced annexin V+/PI- cell increase and DNA fragmentation. There was a correlation between cell apoptosis rates and the concentrations of plumbagin in dose-dependent manner (P < 0.05). It is concluded that for the first time the present study shows that plumbagin can inhibit cell proliferation, block cell cycle and induce apoptosis of APL cell line NB4 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Humans; Leukemia, Promyelocytic, Acute; Naphthoquinones

We have recently purified a tetrameric carbonyl reductase from the cytosolic fraction of pig heart (pig heart carbonyl reductase). Since pig heart carbonyl reductase efficiently reduces all-trans retinal as the endogenous substrate, it probably plays an important role in retinoid metabolism in the heart. The purpose of the present study was to evaluate the inhibitory effects of quinones and flavonoids on the reduction of all-trans retinal to all-trans retinol catalyzed by pig heart carbonyl reductase, using pig heart cytosol. Of quinones tested, 9,10-phenanthrenequinone, a component of diesel exhaust particles, was the most potent inhibitor for the all-trans retinal reduction, and a significant inhibition was also observed for plumbagin and menadione. The order of the inhibitory potencies for flavonoids was kaempferol > quercetin > genistein > myricetin = apigenin = daidzein. However, the inhibitory potencies of flavonoids were much lower than that of 9,10-phenanthrenequinone. 9,10-Phenanthrenequinone competitively inhibited the all-trans retinal reduction, whereas kaempferol exhibited a mixed-type inhibition. It is likely that 9,10-phenanthrenequinone strongly inhibits the reduction of all-trans retinal to all-trans retinol by acting as the substrate inhibitor of pig heart carbonyl reductase present in pig heart cytosol.

Topics: Alcohol Oxidoreductases; Animals; Apigenin; Barbital; Cytosol; Dose-Response Relationship, Drug; Flavonoids; Genistein; Isoflavones; Kaempferols; Kinetics; Molecular Structure; Myocardium; Naphthoquinones; Oxidation-Reduction; Phenanthrenes; Quercetin; Quinones; Retinaldehyde; Swine; Vitamin A; Vitamin K 3

Three 2-naphthaldehydes, including the novel 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde, the simple naphthoquinone plumbagin and the dimeric naphthoquinones diospyrin and 8'-hydroxyisodiospyrin have been isolated from the roots of Diospyrosassimilis and their structures established by spectroscopic analysis.

Topics: Aldehydes; Diospyros; India; Molecular Structure; Naphthalenes; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Plant Roots; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet

The interest of many investigators in naphthoquinones is due to their broad-range of biological actions from phytotoxic to fungicidal. The main aim of this work was to investigate the influence of different pH values of cultivation medium on naphthoquinone content in Dionaea muscipula. For this purpose, we optimized the simultaneous analysis of the most commonly occurring naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin) by high performance liquid chromatography coupled with diode array detector (HPLC-DAD). The most suitable chromatographic conditions were as follows: mobile phase: 0.1 mol l-1 acetic acid:methanol in ratio of 33:67 (%, v/v), flow rate: 0.75 ml min-1 and temperature: 42 degrees C. Moreover, we looked for the most suitable technique for preparation of plant samples (D. muscipula, Juglans regia, Paulownia tomentosa, Impatience glandulifera, Impatience parviflora, Drosera rotundifolia, Drosera spathulata and Drosera capensis) due to their consequent analysis by HPLC-DAD. It clearly follows from the results obtained that sonication were the most suitable technique for preparation of J. regia plants. We also checked the recoveries of the determined naphthoquinones, which were from 96 to 104%. Finally, we investigated the changes in content of plumbagin in D. muscipula plants according to different pH of cultivation medium. The content increased with increasing pH up to 5 and, then, changed gradually. The lower content of plumbagin at lower pH values was of interest to us. Therefore, we determined the content of this naphthoquinone in the cultivation medium, what has not been studied before. We discovered that the lower tissue content of plumbagin was due to secretion of this naphthoquinone into the cultivation medium.

Topics: Chromatography, High Pressure Liquid; Culture Media; Droseraceae; Hydrogen-Ion Concentration; Naphthoquinones; Reproducibility of Results; Spectrophotometry, Ultraviolet

The solubility of 1,4-naphthoquinone, plumbagin, lawsone, and juglone in supercritical carbon dioxide was determined spectroscopically at 40 degrees C, and in the pressure range 8-18 MPa. Their solubilities at 12 MPa were between 0.3 and 10 g L(-1). Plumbagin from Plumbago scandens L. roots was extracted at 40 degrees C and 20 MPa. The extracted plumbagin mass fraction was up to 0.2% in fresh roots but down to about 0.006% in aged roots. n-Hexane and chloroform extraction of such aged roots indicates that the older and dryer the roots are, the stronger they bind plumbagin. Reversed-phase HPLC indicated a relatively pure plumbagin extract with supercritical carbon dioxide.

Topics: Carbon Dioxide; Chromatography, High Pressure Liquid; Kinetics; Molecular Structure; Naphthoquinones; Plant Roots; Plumbaginaceae; Solubility; Spectrum Analysis

The aim of the present study is to develop an automated blood sampling (ABS) method coupled to a liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method to evaluate the oral bioavailability of plumbagin in a conscious freely moving rat. Plumbagin, an herbal ingredient, was isolated from Plumbago zeylanica L. The separation was performed using a reversed phase C18 (150mmx4.6mm I.D.; 5microm) column and was eluted with the mobile phase of water-acetonitrile (40:60, v/v) at a flow-rate of 0.8ml/min. Multiple reaction monitoring (MRM) was used to monitor the transition of the deprotonated molecule m/z 187 [MH](-) to the product ion m/z 159 [MHCO](-) for the plumbagin analysis. The calibration curve was linear over the concentration range of 10-2000ng/ml with a coefficient estimation of 0.995. The intra- and inter-day variations (% relative standard deviation; RSD and % bias) of the assay for rat plasma samples were less than 17%. The limit of detection and the limit of quantification were 5 and 10ng/ml, respectively. Recovery of plumbagin from the rat plasma was about 80%. This LC-MS/MS method has been validated to study the pharmacokinetics of plumbagin in rats. The oral bioavailability (AUC(PO)/Dose(PO))/(AUC(IV)/Dose(IV)) of plumbagin was about 38.7+/-5%.

Topics: Animals; Area Under Curve; Biological Availability; Calibration; Chromatography, High Pressure Liquid; Male; Naphthoquinones; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry

Chemical investigation of the roots of Diospyros assimilis had led to the isolation and characterization of six naphthalene derivatives, two 2-naphthaldehyes, namely 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde 1, 4-hydroxy-5-methoxy-2-naphthaldehye 2, its related isomer 5-hydroxy-4-methoxy-2-naphthaldehyde 3 and three commonly occurring naphthoquinones, diospyrin 4, 8'-hydroxyisodiospyrin 5 and the simple monomer, plumbagin 6. Their chemical structures were established by detailed NMR investigations including 1H and 13C NMR, HSQC, HMBC and NOESY experiments. In addition, the naphthalene derivatives 1-5 were evaluated for their in vitro antiprotozoal activity against protozoan parasites belonging to the genera Trypanosoma, Leishmania and Plasmodium. Among the tested compounds, naphthaldehyde 1 showed moderate inhibition of the growth of the parasites, T. brucei, T. cruzi, L. donovani with IC50 values of 19.82, 12.28 and 38.78 microM and displayed cytotoxicity towards rat skeletal myoblasts (L-6 cells) with IC50 of 174.94 microM, while 2 and 3 were found to be comparatively less active to 1. The dimeric quinones 4 and 5 exhibited good activity against T. brucei and L. donovani with IC50 of 1.12 and 8.82 microM and 12.94 and 16.66 microM respectively.

Topics: Aldehydes; Animals; Antiprotozoal Agents; Cell Survival; Cells, Cultured; Diospyros; Inhibitory Concentration 50; Leishmania donovani; Magnetic Resonance Spectroscopy; Molecular Structure; Naphthalenes; Naphthoquinones; Plant Roots; Rats; Trypanosoma cruzi

This study is the first to investigate the anticancer effect of plumbagin in human breast cancer cells. Plumbagin exhibited cell proliferation inhibition by inducing cells to undergo G2-M arrest and autophagic cell death. Blockade of the cell cycle was associated with increased p21/WAF1 expression and Chk2 activation, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also reduced Cdc2 function by increasing the association of p21/WAF1/Cdc2 complex and the levels of inactivated phospho-Cdc2 and phospho-Cdc25C by Chk2 activation. Plumbagin triggered autophagic cell death but not predominantly apoptosis. Pretreatment of cells with autophagy inhibitor bafilomycin suppressed plumbagin-mediated cell death. We also found that plumbagin inhibited survival signaling through the phosphatidylinositol 3-kinase/AKT signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin, forkhead transcription factors, and glycogen synthase kinase 3beta. Phosphorylation of both of mammalian target of rapamycin downstream targets, p70 ribosomal protein S6 kinase and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased plumbagin-mediated autophagic cell death, whereas reduction of AKT expression by small interfering RNA potentiated the effect of plumbagin, supporting the inhibition of AKT being beneficial to autophagy. Furthermore, suppression of AKT by plumbagin enhanced the activation of Chk2, resulting in increased inactive phosphorylation of Cdc25C and Cdc2. Further investigation revealed that plumbagin inhibition of cell growth was also evident in a nude mouse model. Taken together, these results imply a critical role for AKT inhibition in plumbagin-induced G2-M arrest and autophagy of human breast cancer cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Breast Neoplasms; Cell Division; Cell Growth Processes; Cell Line, Tumor; Female; G2 Phase; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases

To develop a RP-HPLC method to determine plumbagin in Plumbago zeylanica, and to investigate contents of plumbagin in different parts of. P. zeylanica.. The analysis was carried out at 30 degrees C on a Kromasil C18, column eluted with a mobile phase consisting of a mixture of methanol-water (65: 35). The flow rate was 1 mL x min(-1), the detector wavelength was 213 nm.. The calibration curve was linear within the concentration ranges of 0.020 8-0. 104 microg (r = 0. 9999). The average recovery was 98.7%. The contents in the root, stem and leaf were 0.394 5%, 0.050 8%, 0.031 4% respectively.. This method is simple, accurate, replicate and suitable for the determination of plumbagin in P. zeylanica.

Topics: Chromatography, High Pressure Liquid; Naphthoquinones; Plant Leaves; Plant Roots; Plant Stems; Plants, Medicinal; Plumbaginaceae; Quality Control; Reproducibility of Results

Some representative quinones, viz. one naphthoquinone (plumbagin) and five anthraquinones (alizarin, purpurin, chrysazin, emodin, and anthrarufin), were subjected to electrocoagulation. It was found that the rate and extent of coagulation of these compounds appears to correlate with the number and relative position of their phenolic substituent groups, and that all of the coagulated quinones could be recovered. Attempts were then made to electrochemically isolate three quinones, namely plumbagin, morindone and erythrolaccin, from natural sources.

Topics: Anthraquinones; Benzoquinones; Electrocoagulation; Morinda; Naphthoquinones; Pigments, Biological; Plumbaginaceae; Quinones

NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Enzyme Inhibitors; Humans; Kidney Neoplasms; NAD; NADP; NADPH Oxidase 4; NADPH Oxidases; Naphthoquinones; Onium Compounds; Plumbaginaceae; Transfection

A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function sigma(M) protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate sigma(M)-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.

Topics: Anti-Bacterial Agents; ATP-Binding Cassette Transporters; Bacillus subtilis; Bacterial Proteins; Benzene Derivatives; Diamide; Diquat; DNA Mutational Analysis; Drug Resistance, Bacterial; Hydrogen Peroxide; Hydrolases; Mutation; Naphthoquinones; Nitrofurantoin; Paraquat; Pyrogallol; Sigma Factor; tert-Butylhydroperoxide; Viologens; Vitamin K 3

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is an herbal ingredient which is isolated from the root of Plumbago zeylanica L. This herb is a semi-climbing subshrub distributed in thickets or grassland at low elevations of Taiwan. The crushed roots of P. zeylanica L. were ground from lumps to powder and boiled with H2O, 50% EtOH, or 95% EtOH. Chromatographic separation of plumbagin from the herb was carried out using a ZORBAX Extend-C18 column (150 x 4.6 mm I.D.; 5 microm) that was eluted with the mobile phase of water-methanol (10:90, v/v). Multiple reaction monitoring (MRM) was used to monitor the transition of the deprotonated molecule m/z 187 [M-H]- to the product ion m/z 159 [M-H-CO]- for plumbagin analysis. The limit of quantification was determined to and accuracy of 1 ng/ml. Furthermore, the mass fractions of plumbagin in P. zeylanica L. for H2O, 50% EtOH and 95% EtOH were 0.24 +/- 0.04, 3.92 +/- 0.87 and 13.4 +/- 1.59 g/kg, respectively. These results present a reliable liquid chromatography coupled with tandem mass spectrometric (LC-MS/MS) method for the determination of plumbagin form herbal medicines.

Topics: Chromatography, Liquid; Naphthoquinones; Plant Roots; Plumbaginaceae; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization

Candida albicans, the most frequent fungal pathogen of humans, encounters high levels of oxidants following ingestion by professional phagocytes and through contact with hydrogen peroxide-producing bacteria. In this study, we provide evidence that C. albicans is able to coordinately regulate the oxidative stress response at the global cell population level by releasing protective molecules into the surrounding medium. We demonstrate that conditioned medium, which is defined as a filter-sterilized supernatant from a C. albicans stationary-phase culture, is able to protect yeast cells from both hydrogen peroxide and superoxide anion-generating agents. Exponential-phase yeast cells preexposed to conditioned medium were able to survive levels of oxidative stress that would normally kill actively growing yeast cells. Heat treatment, digestion with proteinase K, pH adjustment, or the addition of the oxidant scavenger alpha-tocopherol did not alter the ability of conditioned medium to induce a protective response. Farnesol, a heat-stable quorum-sensing molecule (QSM) that is insensitive to proteolytic enzymes and is unaffected by pH extremes, is partly responsible for this protective response. In contrast, the QSM tyrosol did not alter the sensitivity of C. albicans cells to oxidants. Relative reverse transcription-PCR analysis indicates that Candida-conditioned growth medium induces the expression of CAT1, SOD1, SOD2, and SOD4, suggesting that protection may be mediated through the transcriptional regulation of antioxidant-encoding genes. Together, these data suggest a link between the quorum-sensing molecule farnesol and the oxidative stress response in C. albicans.

Topics: alpha-Tocopherol; Anticoagulants; Antifibrinolytic Agents; Antioxidants; Candida albicans; Culture Media, Conditioned; Ethanol; Farnesol; Fungal Proteins; Gene Expression Regulation, Fungal; Humans; Hydrogen Peroxide; Naphthoquinones; Oxidants; Oxidative Stress; Vitamin K 3

The plant Plumbago zeylanica L. is a semi-climbing shrub that grows throughout Asia and Africa. In our previous study, P. zeylanica L. exhibited high anti-Helicobacter pylori and good bactericidal activities over a wide pH range (pH 2-7). Plumbagin - the major ingredient derived from the roots of P. zeylanica L. - is a naphthoquinone compound. In this study, we investigated plumbagin's anti-H. pylori activity and developed a reversed-phase high-performance liquid chromatography (HPLC) method for quantification of plumbagin from P. zeylanica L. We also observed that plumbagin has strong anti-H. pylori activity, with 0.02-0.16 mg/ml as minimum inhibitory concentrations and 0.16-1.28 mg/ml as minimum bactericidal concentrations. Reversed-phase HPLC was performed with a gradient mobile phase composed of water and methanol, and peaks were detected at 254 nm. Standard curves were linearized in the range of from 10 to 200 microg/ml (regression coefficient r2 = 0.99995). After spikes of 50, 100, and 150 microg/ml of plumbagin standard solution, recovery rates were between 97.45 and 99.24%. Both intra- and inter-day precisions had coefficient variation of less than 1% at concentrations of 50, 100, and 150 microg/ml. The limits of detection and quantitation were 0.02 and 0.06 microg/ml, respectively. Based on validation results, this analytical method is a precise, accurate and stable method to quantify plumbagin derived from P. zeylanica L.

Topics: Chromatography, High Pressure Liquid; Helicobacter pylori; Microbial Sensitivity Tests; Naphthoquinones; Plumbaginaceae; Reproducibility of Results; Sensitivity and Specificity

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.

Topics: Bacterial Proteins; Cation Transport Proteins; Culture Media; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Heat-Shock Response; Hydrogen Peroxide; Lactoperoxidase; Mutation; Naphthoquinones; Oxidative Stress; Salmonella typhimurium

Information available on the mutagenicity of a large number of indigenous drugs commonly employed in the Siddha and Ayurveda systems of medicine is scanty. In this context, the current investigation on plumbagin, 5-hydroxy-2methyl-1,4-napthoquinone, an active principle in the roots of Plumbago zeylanica used in Siddha and Ayurveda for various ailments, was carried out; 16 mg/kg b.w. (LD(50)) was fixed as the maximum dose. Subsequent dose levels were fixed as 50% and 25% of LD(50) amounting to 8 mg and 4 mg/kg b.w., respectively, and given orally for 5 consecutive days in 1% Carboxyl Methyl Cellulose (CMC) to Swiss albino mice weighing 25-30 g. The micronucleus assay was done in mouse bone marrow. Plumbagin was found to induce micronuclei at all the doses studied (4 mg/kg, 8 mg/kg, 16 mg/kg b.w.), and it proves to be toxic to bone marrow cells of Swiss albino mice. Animal treated with cyclophosphamide (40 mg/kg b.w.) served as positive control. In addition, glutathione S-transferase (GST) activity was observed in control, plumbagin (4 mg, 8 mg, 16 mg/kg b.w., respectively), and genotoxin-treated experimental group of animals. No significant change in GST activity was observed with plumbagin dose of 4 mg/kg b.w., whereas GST activity was significantly inhibited by higher doses of plumbagin (8 mg and 16 mg/kg b.w.) and cyclophosphamide.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bone Marrow; Cyclophosphamide; DNA Damage; Female; Glutathione Transferase; Liver; Male; Mice; Micronuclei, Chromosome-Defective; Micronucleus Tests; Naphthoquinones

Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild-type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1-blocked ER-positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1-blocked ER-positive BG-1 ovarian cancer cells. For all three drugs, BRCA1-blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P < 0.05). All three drugs induced loss of mitochondrial membrane potential (DeltaPsi(m)), nuclear condensation, DNA fragmentation, and morphological changes, as observed after 6 h of drug treatment, suggesting apoptosis induction in both BRCA1-blocked and control cells. However, apoptosis induction was greater in BRCA1-blocked cells, the efficacy being in the order of plumbagin > tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 microM in the control cells and 2.68 microM for the BRCA1-blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild-type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1-mutated ovarian cancer patients.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; BRCA1 Protein; Cell Division; Emodin; Enzyme Inhibitors; Estrogen Antagonists; Female; Humans; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Naphthoquinones; Neoplasms, Hormone-Dependent; Ovarian Neoplasms; RNA, Antisense; RNA, Messenger; Tamoxifen; Tumor Cells, Cultured

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

According to the oxidative damage theory a primary cause of aging is the accrual of molecular damage from reactive oxygen species (ROS), particularly superoxide and its derivatives. This predicts that treatments that reduce ROS levels should retard aging. Using the nematode Caenorhabditis elegans, we tested the effects on stress resistance and life span of treatment with EUK-8 and EUK-134, synthetic mimetics of the antioxidant enzyme superoxide dismutase (SOD), which neutralises superoxide. Treatment with SOD mimetics elevated in vivo SOD activity levels, particularly in mitochondria, where up to 5-fold increases in SOD activity were recorded. Treatment with exogenous SOD mimetics did not affect endogenous protein SOD levels. Where life span was reduced by the superoxide generators paraquat and plumbagin, EUK-8 treatment increased life span in a dose-dependent fashion. Yet in the absence of a superoxide generator, treatment with EUK-8 or EUK-134 did not increase life span, even at doses that were optimal for protection against pro-oxidants. Thus, an elevation of SOD activity levels sufficient to increase life span when it is limited by superoxide generators does not retard aging in the absence of superoxide generators. This suggests that C. elegans life span is not normally limited by levels of superoxide and its derivatives.

Topics: Aging; Animals; Antinematodal Agents; Biomimetics; Caenorhabditis elegans; Cyanides; Cytosol; Dose-Response Relationship, Drug; Escherichia coli; Ethylenediamines; Free Radicals; Herbicides; Manganese Compounds; Mitochondria; Naphthoquinones; Organometallic Compounds; Oxidants; Oxygen Consumption; Paraquat; Reactive Oxygen Species; Salicylates; Superoxide Dismutase; Superoxides; Time Factors

There is an emerging evidence that plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) may have potential as a chemotherapeutic agent. However, the growth inhibitory mechanisms of plumbagin have remained unexplored. The aim of the study was to determine whether plumbagin-induced cell death in human cervical cancer cell line, ME-180, exhibited biochemical characteristics of apoptosis and to check whether N-acetyl-l-cysteine (NAC), which is a free radical scavenger, can reverse the cytotoxic effects of plumbagin. It can be concluded from the results that plumbagin inhibits the growth of ME-180 cells in a concentration and time-dependent manner. The cytotoxic effect of plumbagin induced cell death is through the generation of reactive oxygen species (ROS) and subsequent induction of apoptosis as demonstrated by the present data. Treatment of cells with plumbagin caused loss of mitochondrial membrane potential (DeltaPsi(m)), and morphological changes characteristic of apoptosis, such as the translocation of phosphatidyl serine, nuclear condensation, and DNA fragmentation. Moreover, plumbagin-induced apoptosis involved release of mitochondrial cytochrome c and apoptosis inducing factor (AIF), thus activation of caspase-dependent and -independent pathways, as shown by the plumbagin-mediated activation of caspase-3 and -9. Our results also show that pretreatment of ME-180 cells with NAC blocks plumbagin-induced loss of DeltaPsi(m) and subsequent release of cytochrome c, AIF, and caspase-9 and -3 activation, thus inhibiting the apoptotic ability of plumbagin.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspases; Cell Nucleus; Cytochromes c; Dose-Response Relationship, Drug; Female; Humans; Mitochondria; Naphthoquinones; Phosphatidylserines; Reactive Oxygen Species; Time Factors; Uterine Cervical Neoplasms

Bioactivity-directed fractionation of extracts of two Diospyros maritima bark samples from Indonesia,one collected at sea level in a beach forest in Java and the other collected at a slight elevation away from the sea shore on the island of Lombok, yielded a diverse set of secondary metabolites. The naphthoquinone plumbagin (1), although found in extracts of both specimens, constituted a much larger percentage of the former sample, which also yielded a series of plumbagin dimers, maritinone (2), chitranone (3), and zeylanone (4). The latter sample yielded a new naphthoquinone derivative, (4S)-shinanolone (5), and a new natural product coumarin, 7,8-dimethoxy-6-hydroxycoumarin (6), along with three other analogues of plumbagin, 2-methoxy-7-methyljuglone (7), 3-methoxy-7-methyljuglone (8), and 7-methyljuglone (9). The structures of compounds 5 and 6 were elaborated by physical, spectral, and chemical methods. All of the isolates were evaluated in both cytotoxicity and antimicrobial assays, and structure-activity relationships of these naphthoquinones are proposed. Plumbagin (1) and maritinone (2) were evaluated also for in vivo antitumor activity in the hollow fiber assay, but both were found to be inactive.

Topics: Aspergillus niger; Bacteria; Candida albicans; Coumarins; Diospyros; Drug Screening Assays, Antitumor; Humans; Indonesia; KB Cells; Microbial Sensitivity Tests; Naphthoquinones; Plant Bark; Plants, Medicinal; Saccharomyces cerevisiae

The Plumbago genus belongs to the Plumbaginaceae family and it is known due to its variety of biological uses, most of them attributed to the presence of naphthoquinones. Plumbagin is a naturally occurring naphthoquinone that can be obtained from roots of Plumbago scandens L. In order to find out the better technique for plumbagin extraction, were applied: static maceration, dynamic maceration, with assistance of ultrasonic waves and in Soxhlet apparatus. Four compounds were qualitatively detected in all extracts: the naphthoquinones plumbagin and epi-isoshinanolone, palmitic acid and sitosterol. Plumbagin was always the major component in all analyzed extracts and it was quantitatively determined by gas chromatograph coupled with mass spectrometer. Soxhlet was the most efficient extraction technique however, prolonged heating time promoted plumbagin degradation.

Topics: Chromatography, Gas; Naphthoquinones; Plant Extracts; Plant Roots; Plumbaginaceae

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.

Topics: Amidohydrolases; Anti-Bacterial Agents; Bridged Bicyclo Compounds; Cysteine; Dinitrochlorobenzene; Disaccharides; Enzyme Inhibitors; Ethylmaleimide; Gene Deletion; Genes, Bacterial; Genetic Complementation Test; Glycopeptides; Inositol; Iodoacetamide; Mutagenesis, Insertional; Mycobacterium smegmatis; Mycobacterium tuberculosis; Naphthoquinones; Oxidants; Pyrazoles; Rifamycins; Streptomycin; Sulfhydryl Compounds; Vitamin K 3

Plumbago zeylanica (known as "Chitrak") is a useful Indian medicinal plant. The root of the plant and its constituents are credited with potential therapeutic properties including anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective properties. To examine possible mechanisms of action of P. zeylanica (Chitrak), in relation to its reported beneficial properties, antioxidant effects of the aqueous/alcoholic extracts of root, corresponding to medicinal preparations, and the active ingredient, plumbagin, were studied. Methods used included: ferric reducing/antioxidant power (FRAP), radical scavenging of 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS), lipid peroxidation in rat liver mitochondria induced by different agents, and estimating phenolic and flavonoid content. In FRAP/DPPH assays, boiled ethanolic extracts were the most effective, while in the ABTS assay boiled aqueous extracts were the most efficient. These extracts also significantly inhibited lipid peroxidation induced by cumene hydroperoxide, ascorbate-Fe(2+) and peroxynitrite and contained high amounts of polyphenols and flavonoids. To examine the mechanisms of action in detail, antioxidant and pulse radiolysis studies with plumbagin were conducted. The hydroxyl (.OH), alkyl peroxyl (CCl(3)OO.), linoleic acid peroxyl (LOO.), and glutathiyl (GS.) radicals generate a phenoxyl radical upon reaction with plumbagin. The bimolecular rate constants were: .OH, 2.03 x 10(9) dm(3)mol(-1)s(-1); CCl(3)OO., 1.1 x 10(9) dm(3)mol(-1)s(-1); LOO., 6.7 x 10(7) dm(3)mol(-1)s(-1); and GS., 8.8 x 10(8) dm(3)mol(-1)s(-1). In conclusion, our studies reveal that extracts of P. zeylanica and its active ingredient plumbagin have significant antioxidant abilities that may possibly explain some of the reported therapeutic effects.

Topics: Antioxidants; Flavonoids; India; Medicine, East Asian Traditional; Naphthoquinones; Oxidative Stress; Phenols; Phytotherapy; Plant Extracts; Plant Roots; Plumbaginaceae; Spectrophotometry

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

The superoxide generator menadione was previously demonstrated as an inducer of growth stage dependent protein patterns in Mycobacterium tuberculosis. The present study refines this observation by characterizing a novel 27-kDa protein that had not been observed in previous studies relying on younger cultures. A very similar response, based on two-dimensional gel electrophoretic analyses, was induced by the closely related naphthoquinone plumbagin. The 27-kDa protein was also induced by the pro-oxidant peroxisome proliferator gemfibrozil and to a lesser extent by the structurally related compounds fenofibrate and clofibrate. N-terminal sequence data of proteolytic fragments from the 27-kDa protein demonstrated its identity with protein Rv0560c, previously demonstrated to be inducible by salicylate, which also possesses peroxisome proliferating properties. Protein Rv0560c bears three conserved motifs characteristic of S-adenosylmethionine-dependent methyltransferases. Further sequence similarities suggest a function in the bio syn thesis of isoprenoid compounds, e.g., tocopherol, ubiquinone, and sterols. Such involvement is supported by the recognized yet unexplained widespread interference of menadione, salicylate, and fibrates with the isoprenoid quinones ubiquinone, menaquinone, and vitamin K. Induction of Rv0560c by fibrates, salicylate, and naphthoquinones is thus suggested to be caused by action on the plasma membrane, reminiscent of cytochrome P450BM-3 induction by fibrates in Bacillus megaterium, which catalyzes the hydroxylation of fatty acids and thus modulates membrane properties.

Topics: Amino Acid Motifs; Amino Acid Sequence; Bacillus megaterium; Bacterial Proteins; Cell Membrane; Clofibric Acid; Conserved Sequence; Electrophoresis, Gel, Two-Dimensional; Enzyme Activators; Gemfibrozil; Gene Expression Regulation, Bacterial; Genes, Bacterial; Methyltransferases; Molecular Sequence Data; Molecular Weight; Mycobacterium tuberculosis; Naphthoquinones; Sequence Homology, Amino Acid

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.

Topics: Acrylic Resins; Adsorption; Alginates; Calcium Chloride; Cell Culture Techniques; Cells, Cultured; Cells, Immobilized; Chitin; Chitosan; Glucuronic Acid; Hexuronic Acids; Naphthoquinones; Plant Leaves; Plumbaginaceae; Polystyrenes; Sucrose

The effects of plumbagin were investigated on platelet aggregation in vitro and ex vivo, on the binding of thrombin-stimulated platelets to neutrophils, and platelet aggregation induced by intact neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF) activated neutrophils, by use of the methods of Hamburger, McEver and Born, respectively. The results showed that plumbagin in vitro significantly inhibited adenosine diphosphate (ADP)-, arachidonic acid (AA)-, or platelet activating factor (PAF)-induced platelet aggregation, in a concentration-dependent manner. The medium inhibitory concentrations (IC 50 ) were 39.4, 82.7 and 38.1 microM, respectively. Intragastric plumbagin at 10 mg/kg markedly suppressed platelet aggregation induced by ADP, AA, or PAF. Plumbagin decreased the binding between thrombin-stimulated platelets and neutrophils with an IC 50 of 62.9 microM. Plumbagin significantly inhibited washed platelet aggregation stimulated by fMLP- or PAF-activated neutrophils. The IC 50 values were 54.3 and 47.6 microM, respectively. On the other hand, plumbagin and aspirin increased the inhibition of intact neutrophils on AA-induced platelet aggregation. It is suggested that plumbagin inhibited platelet aggregation in vitro and ex vivo, suppressed the binding of activated platelets to neutrophils, inhibited platelet aggregation induced by activated neutrophils, and increased inhibition of intact neutrophils on platelet reactivity. Abbreviations. DMSO:dimethyl sulphoxide fMLP: N-formyl-methionyl-leucyl-phenylalanine ADP:adenosine diphosphate AA:arachidonic acid PAF:platelet activating factor

Topics: Adenosine Diphosphate; Animals; Arachidonic Acid; Blood Platelets; In Vitro Techniques; Male; Naphthoquinones; Neutrophils; Platelet Activating Factor; Platelet Aggregation; Rabbits

Plumbagin is a naturally occurring naphthoquinone isolated from roots of Plumbago scandens. The plant was collected at the Campus of Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. P. scandens is used as a traditional medicine for the treatment of several diseases. The antimicrobial activity of plumbagin was evaluated using the macrodilution method. The compound exhibited relatively specific activity against bacteria and yeast. The minimum inhibitory concentration test showed the growth inhibiton of Staphylococcus aureus at a concentration of 1.56 g/ml and of Candida albicans at a concentration of 0.78 g/ml. These results suggest the naphthoquinone plumbagin as a promising antimicrobial agent.

Topics: Anti-Infective Agents; Candida albicans; Escherichia coli; Microbial Sensitivity Tests; Naphthoquinones; Plant Extracts; Plumbaginaceae; Salmonella typhimurium; Staphylococcus aureus

Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an approximately 200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.

Topics: Antitubercular Agents; Bacterial Proteins; Base Sequence; Clofazimine; DNA, Bacterial; Drug Resistance, Microbial; Isoniazid; Microbial Sensitivity Tests; Molecular Sequence Data; Mycobacterium tuberculosis; Naphthoquinones; Oxidants; Oxidative Stress; Peroxidases; Superoxides

Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polbeta) or DNA polymerase beta (polbeta) gene-knockout (MEFpolbetaKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G(2)/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polbeta-dependent pathway and by blocking both FEN1 and DNA ligase activity in polbeta-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polbeta-dependent and -independent LP-BER pathways in MEF cells.

Topics: Animals; Base Sequence; Cell Cycle; Cell Division; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Polymerase beta; DNA Repair; Embryo, Mammalian; Fibroblasts; Mice; Molecular Sequence Data; Mutation; Naphthoquinones; Proliferating Cell Nuclear Antigen; Purines; Pyrimidines; Reactive Oxygen Species

The effectiveness of the combination of thermosensitive liposomes of plumbagin and hyperthermia is described. Small-sized, thermosensitive liposomes were prepared by thin film hydration and subsequent sonication. The liposomes were characterized for size, phase transition temperature, in vitro drug release and stability. The results of particle size analysis indicated that almost 90% of the vesicles were below 0.19 microm size. The phase transition temperature of the liposomes as determined by differential scanning calorimetry was found to be 41.32 degrees C. The results of in vitro release studies in phosphate buffered saline + mouse plasma indicated that maximum drug release (51.25%) occurred at 42 degrees C compared to the less than 9% release at 37 degrees C. Better stability profile was observed when the plumbagin liposomes were stored at 4 degrees C. When combined with localised hyperthermia (43 degrees C, 30 min or 1 h), liposomal plumbagin administered intravenously to C57BL/6J mice bearing melanoma exhibited better anticancer activity as compared to animals treated with an equivalent dose of free plumbagin with or without hyperthermia, which was evident by enhanced volume doubling time and growth delay.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adjuvants, Immunologic; Algorithms; Animals; Calorimetry, Differential Scanning; Drug Stability; Excipients; Female; Liposomes; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Microscopy, Electron; Naphthoquinones; Particle Size; Temperature

Tropical pitcher plants (Nepenthes) catch animals in their specialized cup-shaped leaves, digest the prey by secreting enzymes, and actively take up the resulting compounds. The benefit of this behaviour is the ability to grow and compete in nutrient-poor habitats. Our present in vitro study shows that not only the nitrogen of alanine fed to the carnivorous organs is used by the plant but that in addition intact C2-units derived from C-2 and C-3 of stable isotope labelled L-alanine serve as building blocks, here exemplarily for the synthesis of the secondary metabolite plumbagin, a potent allelochemical. This result adds a new facet to the benefit of carnivory for plants. The availability of plumbagin by a de novo synthesis probably enhances the plants' fitness in their defence against phytophagous and pathogenic organisms. A missing specific uptake or CoA activation mechanism might be the reason that acetate fed to the pitchers was not incorporated into the naphthoquinone plumbagin. The dihydronaphthoquinone glucosides rossoliside and plumbaside A, here isolated for the first time from Nepenthes, by contrast, showed no incorporation after feeding of any of the two precursors, suggesting these compounds to be storage forms with probably very low turnover rates.

Topics: Alanine; Carbon; Ecology; Food Chain; Glucosides; Magnoliopsida; Naphthoquinones; Sodium Acetate

Plumbagin, a plant-derived bioactive naphthoquinonoid compound, was converted to a hydroquinonoid derivative, which was studied for its tumour-inhibitory and antileishmanial activities for the first time. A similar chemical transformation was undertaken on an analogous dimeric compound, diospyrin, and its bioassay results were compared with those of the plumbagin derivative. Synthesis of the derivative of plumbagin did not result in a marked enhancement of the tumour-inhibitory activity, whereas the improvement was obvious in the case of diospyrin vis à vis its hydroquinonoid analogue. The conversion of diospyrin to the hydroquinonoid compound also led to a substantial increase in the antileishmanial activity, while a similar conversion of plumbagin failed to do so.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antiparasitic Agents; Carcinoma, Ehrlich Tumor; Cell Division; Inhibitory Concentration 50; Leishmania donovani; Male; Mice; Molecular Structure; Naphthoquinones; Phytotherapy; Plant Extracts; Plant Roots; Plumbaginaceae

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instar Helicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Under in vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fern Cheilanthes farinosa enhanced DNA synthesis.

Topics: Animals; DNA; Ecdysone; Ecdysteroids; Ecdysterone; Insect Proteins; Insecticides; Larva; Limonins; Moths; Naphthoquinones; Triterpenes; Wings, Animal

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

A method for the rapid screening of drugs targeting the bioenergetic metabolism of Leishmania spp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels of Leishmania donovani promastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form of Phothinus pyralis luciferase and incubated with free-membrane permeable D-luciferin analogue D-luciferin-[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (DeltaPsi(m)) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 microM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity.

Topics: Adenosine Triphosphate; Animals; Antiprotozoal Agents; Cell Division; Cell Line; Dose-Response Relationship, Drug; Energy Metabolism; Firefly Luciferin; Kinetics; Leishmania donovani; Luciferases; Luminescent Measurements; Membrane Potentials; Mitochondria; Naphthoquinones; Parasitic Sensitivity Tests; Transfection

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 mutagenicity of three nitric oxide (NO) donors, 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite NO and superoxide, diethylamine/NO (DEA/NO) and spermine/NO (SPER/NO), both releasing authentic NO was analyzed using Escherichia coli tester strains IC203, carrying a deletion of the oxyR gene, and its oxyR(+) parent IC188 (the alternative name of WP2 uvrA/pKM101). The OxyR protein is a redox-sensitive transcriptional activator of genes encoding antioxidant enzymes. Strains IC203 and IC188 contain error-prone DNA polymerases polV, encoded by the chromosomal umuDC genes, and polRI, encoded by mucAB genes carried by pKM101. SIN-1 was determined to be an oxidative mutagen giving a positive response only in IC203, whereas DEA/NO and SPER/NO induced similar positive responses in IC203 and IC188 and were considered as non-oxidative mutagens. The spectrum of ochre suppressors in Trp(+) revertants induced by SIN-1 in IC203 was characterized by a higher number of TA-->AT transversions and GC-->AT transitions, and a lower number of GC-->TA transversions, with respect to the untreated control. The mutagenicity of SIN-1 in IC203, probably induced by peroxynitrite through reactive derivatives, was enhanced in the presence of plumbagin (PLB), a superoxide generator. Superoxide generation by PLB, as well as formation of peroxynitrite in cells treated with SIN-1, evaluated by monitoring the oxidation, respectively, of dihydroethidium and dihydrorhodamine 123, were greater in IC203 than in IC188. Formation of peroxynitrite in IC203 treated with SIN-1 was stimulated by PLB. After treatment with DEA/NO and SPER/NO the number of revertants scored in IC188 was higher than in strains IC187, containing only polV, and IC204, deficient in both polV and polRI. For these compounds, induced suppressor revertants in IC187 and IC204 were almost exclusively GC-->AT transitions, whereas in IC188 significant levels of GC-->TA and TA-->AT transversions were also induced. Mutagenesis by both DEA/NO and SPER/NO was partially inhibited in the presence of PLB. The results show the usefulness of the new tester strain IC203 to differentiate NO-promoted mutagenic mechanisms that involve or do not involve oxygen radicals.

Topics: Diethylamines; DNA Damage; Escherichia coli; Genes, Bacterial; Genes, Suppressor; Models, Genetic; Molsidomine; Mutagenicity Tests; Mutagens; Naphthoquinones; Nitric Oxide Donors; Spermine; Superoxides

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

Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.

Topics: Animals; Antimalarials; Cytochrome c Group; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Kinetics; Mice; Mitochondria, Liver; Molecular Sequence Data; Naphthoquinones; Oxidation-Reduction; Oxygen; Plasmodium falciparum; Succinate Dehydrogenase; Succinic Acid; Thenoyltrifluoroacetone; Ubiquinone

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

Several species of the genus Candida decode the standard leucine CUG codon as serine. This and other deviations from the standard genetic code in both nuclear and mitochondrial genomes invalidate the notion that the genetic code is frozen and universal and prompt the questions 'why alternative genetic codes evolved and, more importantly, how can an organism survive a genetic code change?' To address these two questions, we have attempted to reconstruct the early stages of Candida albicans CUG reassignment in the closely related yeast Saccharomyces cerevisiae. These studies suggest that this genetic code change was driven by selection using a molecular mechanism that requires CUG ambiguity. Such codon ambiguity induced a significant decrease in fitness, indicating that CUG reassignment can only be selected if it introduces an evolutionary edge to counteract the negative impact of ambiguity. We have shown that CUG ambiguity induces the expression of a novel set of stress proteins and triggers the general stress response, which, in turn, creates a competitive edge under stress conditions. In addition, CUG ambiguity in S. cerevisiae induces the expression of a number of novel phenotypes that mimic the natural resistance to stress characteristic of C. albicans. The identification of an evolutionary advantage created by CUG ambiguity is the first experimental evidence for a genetic code change driven by selection and suggests a novel role for codon reassignment in the adaptation to new ecological niches.

Topics: Adaptation, Biological; Arsenites; Blotting, Northern; Cadmium Chloride; Candida; Cell Survival; Codon; Cycloheximide; Dose-Response Relationship, Drug; Enzyme Inhibitors; Evolution, Molecular; Genetic Code; Genetic Variation; Heat-Shock Proteins; Hydrogen Peroxide; Hydrogen-Ion Concentration; Leucine; Models, Biological; Naphthoquinones; RNA, Transfer; Saccharomyces cerevisiae; Serine; Sodium Chloride; Sodium Compounds; Superoxide Dismutase; Temperature

MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. In this study, it was demonstrated that sodium salicylate, plumbagin, 2, 4-dinitrophenol, and menadione-inducers of the marRAB operon in whole cells-all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.

Topics: 2,4-Dinitrophenol; Antineoplastic Agents, Phytogenic; Bacterial Proteins; Drug Resistance, Microbial; Escherichia coli; Escherichia coli Proteins; Naphthoquinones; Operon; Plasmids; Repressor Proteins; Restriction Mapping; Sodium Salicylate; Vitamin K

We have used two hydroxylated naphthoquinol menaquinol analogues, reduced plumbagin (PBH2, 5-hydroxy-2-methyl-1,4-naphthoquinol) and reduced lapachol [LPCH2, 2-hydroxy-3-(3-methyl-2-butenyl)-1, 4-naphthoquinol], as substrates for Escherichia coli anaerobic reductases. These compounds have optical, solubility and redox properties that make them suitable for use in studies of the enzymology of menaquinol oxidation. Oxidized plumbagin and oxidized lapachol have well resolved absorbances at 419 nm (epsilon=3.95 mM-1. cm-1) and 481 nm (epsilon=2.66 mM-1.cm-1) respectively (in Mops/KOH buffer, pH 7.0). PBH2 is a good substrate for nitrate reductase A (Km=282+/-28 microM, kcat=120+/-6 s-1) and fumarate reductase (Km=155+/-24 microM, kcat=30+/-2 s-1), but not for DMSO reductase. LPCH2 is a good substrate for nitrate reductase A (Km=57+/-35 microM, kcat=68+/-13 s-1), fumarate reductase (Km=85+/-27 microM, kcat=74+/-6 s-1) and DMSO reductase (Km=238+/-30 microM, kcat=191+/-21 s-1). The sensitivity of enzymic LPCH2 and PBH2 oxidation to 2-n-heptyl-4-hydroxyquinoline N-oxide inhibition is consistent with their oxidation occurring at sites of physiological quinol binding.

Topics: Anaerobiosis; Binding Sites; Electrochemistry; Electron Transport Complex IV; Enzyme Inhibitors; Escherichia coli; Hydroxyquinolines; Kinetics; Molecular Structure; Naphthoquinones; Oxidoreductases; Spectrophotometry; Substrate Specificity

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

We have identified an RNA polymerase sigma factor, sigmaR, that is part of a system that senses and responds to thiol oxidation in the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding sigmaR caused sensitivity to the thiol-specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of sigmaR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5-6 bp. trxBp1 is transiently induced at least 50-fold in response to diamide treatment in a sigR-dependent manner. Purified sigmaR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for sigmaR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR-null mutant, and purified sigmaR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70-fold) following treatment with diamide. We propose a model in which sigmaR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, sigmaR activity is switched off, resulting in down-regulation of trxBA and sigR. We present evidence that the sigmaR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Diamide; Gene Expression Regulation, Bacterial; Genes, Bacterial; Homeostasis; Models, Genetic; Molecular Sequence Data; Mutation; Naphthoquinones; Oxidative Stress; Promoter Regions, Genetic; Sigma Factor; Signal Transduction; Streptomyces; Sulfhydryl Compounds; Thioredoxin-Disulfide Reductase; Thioredoxins; Transcription, Genetic; Vitamin K

The effect of plumbagin, a naphthoquinone from the roots of the Indian medicinal plant Plumbago rosea, and Cobalt-60 gamma radiation was studied on Ehrlich ascites carcinoma in vivo, taking cytogenetic damage and cell cycle changes as experimental endpoints. Plumbagin (5 mg/kg body wt, P1) administered intraperitoneally produced a significant increase in the percentage of S-phase as well as G2-M cells with a corresponding decrease in the G1 phase at different post-treatment times. Radiation (7.5 Gy, RT) alone produced the classical G2 block at 1 hr, which persisted with a continuous increase throughout the post-treatment observation period. The combination treatment produced a similar effect as that of RT on G2-M cells, but its effect on the G1 phase was more pronounced than the latter. While P1 treatment produced a small increase in the percentage of labeled S-phase cells, combination treatment significantly reduced the labeled S-phase cells with a corresponding increase in the unlabeled fraction. Drug or radiation alone significantly increased micronuclei induction at various post-treatment times and the combination of the two further enhanced this effect additively. The mechanism of interaction of P1 with radiation in bringing about this effect is not clear.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Ehrlich Tumor; Cell Cycle; Cytogenetics; Female; Male; Mice; Micronucleus Tests; Naphthoquinones; Radiation-Sensitizing Agents

Addition of hydrogen peroxide, menadione, or plumbagin to growing cultures of the fission yeast Schizosaccharomyces pombe increased trehalase activity. The effect was inhibited only slightly in the presence of cycloheximide, indicating that the stimulation of trehalase triggered by oxidative stress is mostly due to posttranscriptional activation. Northern blot analysis of trehalase mRNA level revealed that oxidative stress also induces a moderate rise in transcription of trehalase. Mutants disrupted in genes encoding elements of the mitogen-activated protein kinase (MAPK) cascade showed a reduced increase in trehalase activity upon oxidative challenge, which was coincident with a block in transcription of trehalase. Taken together, the results support the idea that the enhancement of trehalase by oxidative stress is due to enzyme activation (via the Pka1/Sck1 phosphorylation pathway) and induction of trehalase mRNA (via the MAPK signaling pathway). In spite of the trehalase increase, a net accumulation of trehalose was noticed during the oxidative stress.

Topics: Cycloheximide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Hydrogen Peroxide; Naphthoquinones; Oxidative Stress; RNA Processing, Post-Transcriptional; RNA, Messenger; Schizosaccharomyces; Transcription, Genetic; Trehalase; Vitamin K

To investigate the chemical constituents of the whole plant of Drosera peltata var. lunata collected in Tibet.. Compounds were separated by means of pH gradient extraction and rechromatography on silica gel, and the structures were determined by spectral analysis and physicochemical evidence.. Five crystalline compounds were obtained and elucidated as plumbagin(1), droserone(2), hydroxydroserone(3), beta-sitosterol(4) and stigmasterol(5) respectively.. Compound 3 and 5 were isolated from the plant for the first time.

Topics: Drosera; Drugs, Chinese Herbal; Molecular Structure; Naphthoquinones; Plant Extracts; Plants, Medicinal; Stigmasterol

Inclusion complex of plumbagin was prepared with betacyclodextrin employing neutralization method. The toxicity of the drug was reduced and the antitumor efficacy was enhanced on complexation with betacyclodextrin.

Topics: Absorption; Analysis of Variance; Animals; Antineoplastic Agents, Phytogenic; beta-Cyclodextrins; Carcinogens; Carcinoma, Ehrlich Tumor; Cyclodextrins; Dose-Response Relationship, Drug; Drug Carriers; Drug Synergism; Female; Lethal Dose 50; Mice; Mice, Inbred BALB C; Naphthoquinones; Neoplasm Transplantation

Withaferin A (WA), a steroidal lactone, and Plumbagin (Pl), a naphthoquinone, from the roots of Withania somnifera and Plumbago rosea, respectively, have been shown to possess growth inhibitory and radiosensitizing effects on experimental mouse tumours. An aqueous extract of the leaves of Ocimum sanctum (OE) was found to protect mice against radiation lethality. Therefore, the radiomodifying effects of the above plant products on the bone marrow of the adult Swiss mouse was studied. Single doses of WA (30 mg kg-1) or Pl (5 mg kg-1) were injected intraperitoneally (ip) and OE (10 mg kg-1) was injected ip once daily for five consecutive days. Administration of extracts was followed by 2 Gy whole body gamma irradiation. Bone marrow stem cell survival was studied by an exogenous spleen colony unit (CFU-S) assay. The effects of WA and Pl were compared with that of cyclophosphamide (CP) and radioprotection by OE was compared with that of WR-2721 (WR). Radiation reduced the CFU-S to less than 50% of normal. WA, CP and Pl significantly enhanced this effect and reduced the CFU-S to almost the same extent (to < 20% of normal), although individually WA and Pl were less cytotoxic than CP. These results indicate that radiosensitization by WA and Pl is not tumour specific. OE significantly increased CFU-S compared with radiotherapy (RT) alone. OE+RT gave a higher stem cell survival (p < 0.05) than that produced by WR+RT. While WR alone had a toxic effect, OE treatment showed no such effect, suggesting that the latter may have an advantage over WR in clinical application.

Topics: Animals; Bone Marrow; Cell Survival; Ergosterol; Female; Hematopoietic Stem Cells; Male; Mice; Naphthoquinones; Plant Extracts; Radiation-Protective Agents; Radiation-Sensitizing Agents; Whole-Body Irradiation; Withanolides

With a view to increase efficiency and reduce toxicity of Plumbagin, an attempt was made to formulate plumbagin as a controlled release preparation using various carriers and test for their antitumor and antifertility activities. Niosomes and albumin microspheres were used as carriers. In vitro data showed promising results for these formulations thus they were taken up for in vivo assessment. Given at a dose of 5 mg/kg, ip the albumin microspheres showed promising antitumor and antifertility activity when compared to the niosomes on control. Animal survival data also indicated slight improvement in survival rate and thus antitumoral activity. Also, an interesting point was that the antifertility activity was affected through an antiovulatory action as seen from histopathological studies.

Topics: Animals; Antineoplastic Agents, Phytogenic; Contraceptive Agents; Delayed-Action Preparations; Drug Carriers; Female; Fertility; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Microspheres; Naphthoquinones; Ovary; Pregnancy; Rats

We isolated a promoter that is inducible by paraquat, a superoxide-generating agent, from Escherichia coli using the promoter-probe plasmid pRS415. Sequence analysis revealed that the promoter derives from the ribA gene encoding GTP cyclohydrolase II, which is the first enzyme in the biosynthetic pathway of riboflavin. We fused the lacZ gene with the ribA promoter to monitor the expression of the gene in the single-copy state. LacZ expression from the ribA promoter was induced about eight-fold by 200 microM paraquat. Other known superoxide generators, menadione and plumbagin, also induced the expression of beta-galactosidase in the fusion strain. On the other hand, no significant induction was observed following treatment with hydrogen peroxide, ethanol or heat shock. Induction of beta-galactosidase was significantly reduced by the introduction of a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that the ribA gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis and putative binding sites for SoxS in both orientations were identified. GTP cyclohydrolase II activity in soluble extracts of E. coli increased more than three-fold on treatment with paraquat. This increase was dependent on the soxRS locus, and reflects the increase in transcript levels. However, flavin pools did not change significantly. A possible role for ribA induction during superoxide stress is discussed.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; beta-Galactosidase; Binding Sites; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; GTP Cyclohydrolase; Molecular Sequence Data; Naphthoquinones; Oxidative Stress; Paraquat; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulon; RNA, Bacterial; RNA, Messenger; Trans-Activators; Transcription Factors; Vitamin K

In electrically driven guinea pig left atria, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; 0.5-10 microM) produced a marked positive inotropic effect that was about 65% that caused by isoprenaline in the same experimental conditions. The effect was mainly not dependent on catecholamine release from adrenergic stores. An EC50 of 3 microM was calculated from the concentration-response curves. The increase in force of contraction was followed by a nonreversible contracture. Plumbagin was reduced by cardiac mitochondrial and soluble reductases with consequent generation of large amounts of superoxide anion. The assay of reduced glutathione/oxidized glutathione content in atria, treated with 10 microM plumbagin and frozen at the appearance of increase in diastolic tension, showed a significant decrease in reduced glutathione (-52% with respect to control atria) and a 5-fold increase in oxidized glutathione levels. Moreover, in the same experimental conditions a significant decrease in adenosine triphosphate (-55% with respect to the controls) and in adenylate energy charge (from 0.92-0.64) was observed. Of the enzymes and transport systems involved in the control of the cardiac contractility, the sarcoplasmic reticulum Ca2+ pump seemed to be a specific target for plumbagin. After 30 min of incubation with cardiac sarcoplasmic reticulum membrane vesicles, plumbagin inhibited Ca2+ uptake by the pump in a concentration-dependent manner (IC50 = 3 microM). On the basis of these results, the increase in diastolic tension caused by plumbagin appears to be related to intracellular Ca2+ accumulation, due both to the low availability of adenosine triphosphate for ionic pumps and direct inhibition of Ca2+ reuptake in sarcoplasmic reticulum.

Topics: Animals; Cardiotonic Agents; Dose-Response Relationship, Drug; Guinea Pigs; Heart Atria; Isoproterenol; Naphthoquinones

Plumbagin niosome were prepared using a lipid layer hydration method, and drug entrapment was measured. The acute toxicity studies were conducted following treatment with free and niosomal plumbagin. The antitumour activity of niosomal plumbagin in a solid tumor (sarcoma-180) and Ehrlich ascites model was evaluated. Niosome-encapsulated plumbagin was less toxic than free drug. The antitumour activity of the drug was also better after encapsulation. The better anticancer activity can be justified with the help of LD50 survival studies and study of tumour volume doubling time.

Topics: Animals; Antineoplastic Agents, Phytogenic; Capsules; Carcinoma, Ehrlich Tumor; Chemistry, Pharmaceutical; Dose-Response Relationship, Drug; Drug Carriers; Drug Screening Assays, Antitumor; Liposomes; Mice; Mice, Inbred BALB C; Naphthoquinones; Sarcoma 180; Surface-Active Agents

Topical treatment of the phytochemical plumbagin in doses ranging 0.005-5 micrograms prevented oocyte development and affected fecundity and fertility in M. domestica. The treatment to wandering larvae was less effective as the compound could only effect the fertility to a significant level whereas the fecundity was not significantly reduced. The effect of the compound was more pronounced in adult treatments where both fecundity and fertility reduced drastically. The compound also effected the oocyte maturation as it arrested the development of vitellogenic oocyte at stage six. As the juvenile hormone analogue methoprene and moulting hormone 20-hydroxyecdysone or the mixture of these hormones could not restore the development of the oocyte in ovaries of plumbagin treated flies, it is concluded that the compound does not effect the female houseflies through hormonal pathways, instead in all probability it acts like a cytotoxic compound.

Topics: Animals; Chemosterilants; Female; Gonadotropins; Houseflies; Naphthoquinones; Pest Control, Biological

Mouse melanoma cells were treated with plumbagin, a naphthoquinone, from the plant Plumbago rosea at 0.5 microgram/ml (PI) for 60 min either alone or followed by 2 Gy gamma radiation (RT). Response to the different treatments was assessed by following the cell growth up to 5 days post treatment. PI alone produced a significant decrease in the cell count on days 3 and 4, whereas RT treatment significantly enhanced the growth inhibitory effect when compared to RT or PI alone. These findings suggests the radiosensitizing effect of PI on mouse melanoma cells in vitro, supporting the earlier in vivo findings.

Topics: Animals; Cell Division; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Naphthoquinones; Radiation-Sensitizing Agents; Tumor Cells, Cultured

The effect of aging on the ability of Caenorhabditis elegans to adapt to oxidative stress was examined. Oxidative stress was applied with the quinone plumbagin or with hyperoxia, both of which are expected to increase intraorganismal production of O2.- and of H2O2. Young nematodes adapted by increasing their content of superoxide dismutase (SOD) and they survived, whereas older nematodes did not induce superoxide dismutase and suffered loss of viability. It thus appears that, in C. elegans, loss of adaptability to oxidative stress, monitored in terms of induction of SOD, is a hallmark of senescence.

Topics: Adaptation, Physiological; Aging; Animals; Antinematodal Agents; Caenorhabditis elegans; Hydrogen Peroxide; Kinetics; Naphthoquinones; Oxidative Stress; Oxygen; Superoxide Dismutase; Superoxides

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

The modulatory ability of plumbagin, a natural product from Plumbago zeylanica, was studied on peritoneal macrophages of BALB/c mice. The macrophage functions evaluated were bactericidal activity, hydrogen peroxide and superoxide anion release. The bactericidal capacity of in vivo plumbagin-treated mouse macrophages was estimated against Staphylococcus aureus. In low doses plumbagin exerted a constant increase in bactericidal activity throughout the study period whereas with a high dose a higher response was observed up to six weeks. But in the next two weeks a considerable decline in the bactericidal activity was noticed compared to low dose. Plumbagin was also seen to exert a similar response on oxygen radical release by macrophages in vivo showing a clear correlation between oxygen radical release and the bactericidal activity. The data indicate that plumbagin augments the macrophage bactericidal activity by potentiating the oxyradical release at low concentration whereas at the higher concentration it has inhibitory activity.

Topics: Adjuvants, Immunologic; Animals; Female; Hydrogen Peroxide; Macrophages; Mice; Mice, Inbred BALB C; Naphthoquinones; Staphylococcus aureus; Superoxides

Nine mutants isolated from CHO.K1 cells with increased sensitivity to the lethal effect of plumbagin (PG), a powerful superoxide generator, were classified into five groups, A-E, according to their sensitivity to PG and methyl viologen (MV). Two mutants of group B (Pa13 and Pb4) were sensitive to both drugs, and two mutants of group C (Pa14 and Pa15) were moderately sensitive to PG and extremely sensitive to MV. To mitomycin C (MMC) these mutants showed cross-sensitivity; especially Pa13 and Pb4 (group B) were highly sensitive to MMC. Genetic complementation analyses of these four mutants were carried out using MV sensitivity. Sensitivity group B was divided into two complementation group, I and II. Pa14 and Pa15 belonged to the same complementation group III. These four mutants were also classified into three complementation groups for MMC sensitivity. Because Pa13 and Pb4 were also sensitive to cis-diamminedichloroplatinum(II), they may have a defect in the repair of DNA crosslinks induced by these agents. A complementation group IV (Pa2 and Pa8) was also suggested based on the studies of MMC sensitivity.

Topics: Animals; Antineoplastic Agents; Cell Survival; CHO Cells; Cricetinae; Genetic Complementation Test; Mutation; Naphthoquinones; Paraquat; Reactive Oxygen Species

Exponentially growing Saccharomyces cerevisiae cells are more sensitive to oxidants such as hydrogen peroxide and superoxides than stationary phase cells. Using disruption mutations in the genes encoding the two S. cerevisiae superoxide dismutases, we show that the principal mechanism of toxicity of redox-cycling compounds, such as menadione and plumbagin, is via the production of superoxide anions. Using two-dimensional polyacrylamide gel electrophoresis we have compared the pattern of protein expression in cells labelled with L-[35S]methionine and stressed with either H2O2 or menadione. Three groups of proteins were evident: those whose levels are elevated by both H2O2 and menadione, and those specifically induced by either H2O2 or menadione. Experiments with promoter fusions demonstrated that one of the heat inducible forms of HSP70 (SSA1) was inducible with H2O2. Furthermore, induction of the yeast H2O2-responsive TRX2 promoter by menadione required the metabolism of menadione.

Topics: Base Sequence; Cell Cycle; DNA Primers; DNA, Fungal; Gene Expression Regulation, Fungal; Genes, Fungal; Hydrogen Peroxide; Molecular Sequence Data; Mutagenesis; Naphthoquinones; Oxidative Stress; Saccharomyces cerevisiae; Superoxide Dismutase; Superoxides; Vitamin K

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 x 10(-6) recepient-1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 alpha cells at a frequency of 1 x 10(-8) recepient-1. The growth rate of E. coli DH5 alpha (pUPI199) was slower as compared with E. coli DH5 alpha. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 alpha (pUPI199) effluxed 63% of the accumulated silver ions.

Topics: Acinetobacter; Anti-Bacterial Agents; Biological Transport; Conjugation, Genetic; DNA, Bacterial; Drug Resistance, Microbial; Escherichia coli; Metals; Naphthoquinones; R Factors; Silver Nitrate

Polyphenols have been implicated in the virulence and oxidant resistance of Cryptococcus neoformans. Although monomeric polyphenols did not protect against the prooxidant, plumbagin, polymeric dopamine-melanin conferred resistance both to hypochlorite and to permanganate. The physiologic antioxidant capacity conferred by melanin was found to be 21.3 x 10(-15) mole-equivalents per cell, a value which approximates oxidant production by stimulated macrophages.

Topics: Antioxidants; Asparagine; Cryptococcus neoformans; Dopamine; Hypochlorous Acid; Kinetics; Melanins; Microbial Sensitivity Tests; Naphthoquinones; Oxidants; Phenotype; Potassium Permanganate; R Factors

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) isolated from Plumbago zeylanica Linn, when administered orally, at a dosage of 4 mg/kg body weight induces tumour regression in 3-methyl-4-dimethyl aminoazobenzene (3MeDAB) induced hepatoma in Wistar male rats. The purpose of this investigation was to identify the changes in the rate of glycolysis and gluconeogenesis in tumour-bearing rats and the effects of treatment with Plumbagin. The levels of certain glycolytic enzymes, namely, hexokinase; phosphoglucoisomerase; and aldolase levels increased (p < 0.001) in hepatoma bearing rats, whereas they decreased in Plumbagin administered rats to near normal levels. Certain gluconeogenic enzymes, namely, glucose-6-phosphatase and fructose-1,6-diphosphatase decreased (p < 0.001) in tumour hosts, whereas Plumbagin administration increased the gluconeogenic enzyme levels in the treated animals. These investigations indicate the molecular basis of the different biological behaviour of 3MeDAB induced hepatoma and the anticarcinogenic property of Plumbagin against hepatoma studied in rats.

Topics: Animals; Antineoplastic Agents, Phytogenic; Fructose-Bisphosphatase; Fructose-Bisphosphate Aldolase; Gluconeogenesis; Glucose; Glucose-6-Phosphatase; Glucose-6-Phosphate Isomerase; Glycolysis; Hexokinase; Liver Neoplasms, Experimental; Male; Naphthoquinones; Rats; Rats, Wistar

Redox-cycling agents catalyze the flow of reducing equivalents to oxygen; this process generates superoxide ion and other reduced oxygen species. Measurements of redox-cycling activity have been performed previously by studying cyanide-resistant oxygen consumption (respiration) of Escherichia coli cells. E. coli strain GK100, lacking both terminal oxidases, has almost no measurable respiration. We show that the use of this strain eliminates the requirement for cyanide in measurements of redox-cycling activity. The addition of either menadione sodium bisulfite or plumbagin, well-known redox-cycling agents, to GK100 cells resulted in high levels of oxygen consumption. The rate of menadione bisulfite-induced oxygen consumption in this respiration-deficient strain, in the absence of cyanide, was comparable to the cyanide-resistant respiration of isogenic respiration-proficient E. coli strains. In GK100 cells, cyanide increased menadione bisulfite-induced oxygen consumption but had no effect on plumbagin-induced oxygen consumption.

Topics: Cyanides; Cytochrome b Group; Cytochrome d Group; Cytochromes; Electron Transport; Escherichia coli; Escherichia coli Proteins; Mutation; Naphthoquinones; Oxidation-Reduction; Oxygen Consumption; Sulfites; Vitamin K

Plumbagin, 3,3'-biplumbagin and 8,8'-biplumbagin are naphthoquinones isolated by activity-directed fractionation from a Bolivian plant, Pera benensis, used in folk medicine as treatment of cutaneous leishmaniasis caused by Leishmania braziliensis. BALB/c mice were infected with L. mexicana or L. venezuelensis and treated 24 h after the parasitic infection with plumbagin (5 or 2.5 mg/kg/day), 3,3'-biplumbagin, 8,8'-biplumbagin (25 mg/kg/d) or Glucantime (200 mg/kg/d). Lesion development was the criteria employed to evaluate the inhibitory effect. The bis-naphthoquinones were less potent than Glucantime against L. amazonensis and L. venezuelensis. Plubagin and Glucantime delayed the development of L. amazonensis and L. venezuelensis. Assays of a single local treatment on foot-pad infection two weeks after the parasitic inoculation with L. amazonensis showed that 8,8'-biplumbagin (50 mg/kg/d) was as potent as Glucantime (400 mg/kg/d).

Topics: Animals; Antiprotozoal Agents; Disease Models, Animal; Female; Leishmaniasis, Cutaneous; Leishmaniasis, Mucocutaneous; Male; Meglumine; Meglumine Antimoniate; Mice; Mice, Inbred BALB C; Naphthoquinones; Organometallic Compounds; Plant Extracts

The ability of naphthoquinones to generate reactive oxygen species has been widely exploited in studies of oxidative stress. However, excess superoxide dismutase and catalase failed to protect Escherichia coli in rich medium against growth inhibition by plumbagin, indicating that its toxic effect was not due to the production of partially reduced oxygen species. Respiration failed immediately upon the addition of growth-inhibitory levels of plumbagin. Studies in vitro showed that plumbagin and other redox-active quinones intercept electrons from NADH dehydrogenase, the primary respiratory dehydrogenase in glucose-containing media. An excess of oxidative substrate, such as plumbagin, inactivates this enzyme, which appears to be redox-regulated. The resultant respiratory arrest is a cautionary example of metabolic dysfunction from redox-cycling drugs that cannot be attributed to superoxide or hydrogen peroxide.

Topics: Cell Membrane; Cytochrome c Group; Cytosol; Escherichia coli; Hydrogen Peroxide; Kinetics; NADH Dehydrogenase; Naphthoquinones; Oxidation-Reduction; Oxygen Consumption; Quinones; Structure-Activity Relationship; Superoxide Dismutase; Superoxides

Plumbagin and shikonin, plant metabolites which have naphthoquinone structures, induced mammalian topoisomerase II-mediated DNA cleavage in vitro. Treatment of a reaction mixture containing these naphthoquinones and topoisomerase II at an elevated temperature (65 degrees C) resulted in a great reduction in DNA cleavage, suggesting that the mechanism of the topoisomerase II-mediated DNA cleavage induced by these naphthoquinones is through formation of a cleavable complex, as seen with antitumor agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide and demethylepipodophyllotoxin ethylidene-beta-glucoside. Lawson and lapacol, which are structurally related plant metabolites with naphthoquinone moieties, could not induce topoisomerase II-mediated DNA cleavage. Plumbagin and shikonin induced a similar DNA cleavage pattern with topoisomerase II which was different from the cleavage patterns induced with other known topoisomerase II-active drugs. A DNA-unwinding assay with T4 DNA ligase showed that shikonin, lawson, and lapacol did not intercalate into DNA, while plumbagin and 2-methyl-1,4-naphthoquinone intercalate into DNA, but to a lower degree than 4'-(9-acridinylamino)methanesulfon-m-anisidide does.

Topics: Animals; Antineoplastic Agents, Phytogenic; DNA Damage; DNA Topoisomerases, Type II; Drug Synergism; Enzyme Induction; Intercalating Agents; Mice; Mice, Inbred BALB C; Naphthoquinones

This paper reports the antimutagenic activity of plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) against certain known chemical mutagens in a standard mutagenicity test system of Ames using S. typhimurium strains. Plumbagin by itself did not show any mutagenic effect, whereas it reduced significantly the mutagenic effect of 4-nitrophenylene diammine, phenyl hydrazine and sodium azide in test strains of S. typhimurium, suggesting that plumbagin possessed antimutagenic activity.

Topics: Azides; Humans; Indicators and Reagents; Mutagenesis; Mutagenicity Tests; Naphthoquinones; Phenylhydrazines; Salmonella typhimurium; Sodium Azide

Topics: Bacillus subtilis; Ethanol; Hot Temperature; Hydrogen Peroxide; Naphthoquinones; Paraquat

1. Plumbagin (5-hydroxy,2-methyl-1,4-napthoquinone) inhibited the motility and survival of Haemonchus contortus first-stage larvae (L1) with an ED50 of 1 microgram/ml, but was less effective in preventing the development of H. contortus to infective third-stage larvae in a faecal slurry assay. 2. Of the structural analogs tested, plumbagin was the most potent in preventing development of L1 followed in decreasing order of potency by 1,4-naphthoquinone, 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone. 3. Plumbagin had a biphasic effect on development of the fourth-stage Ascaris suum larvae that caused an increase in growth at low concentrations but was lethal at higher doses. 4. Plumbagin and 1,2-napthoquinone partially inhibited embryonation of A. suum eggs.

Topics: Animals; Antinematodal Agents; Ascaris; Feces; Female; Haemonchus; Naphthoquinones; Rabbits; Sheep

A comparative study was made of the susceptibility of broad-host range vector plasmids belonging to Inc P1 and Q groups in Escherichia coli to various curing agents. Plumbagin and SDS eliminated RP4 (Inc P1 group) plasmid whereas pKT231 (Inc Q) and pRK2013 (having ColE1 replicon) were eliminated by hexamine ruthenium (III) chloride, alpha-santonin, coumermycin A1 and cis-dichloro diamine platinum (II). The curing activity of these agents was specific.

Topics: Aminocoumarins; Anti-Bacterial Agents; Cisplatin; Coumarins; Escherichia coli; Genetic Vectors; Naphthoquinones; Plasmids; Ruthenium; Ruthenium Compounds; Santonin; Sodium Dodecyl Sulfate

Plumbagin, an active principle from Plumbaginaceous plants, produced a triphasic inotropic response (first positive, second negative, and third positive phases) in guinea-pig papillary muscle. The inotropic potency of plumbagin at the first positive phase was pD2 6.40, and the methylation of 5-hydroxy group of plumbagin reduced the potency. The triphasic pattern of inotropism of plumbagin was unaffected by reserpine or propranolol treatments. Plumbagin did not produce the positive inotropic effects under anoxic conditions or in the presence of mitochondrial oxidative phosphorylation inhibitors such as 2,4-dinitrophenol and dicumarol.

Topics: Animals; Cardiotonic Agents; Female; Guinea Pigs; In Vitro Techniques; Male; Naphthoquinones; Papillary Muscles

lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.

Topics: Amino Acid Sequence; Biological Transport; Cell Membrane; Cysteine; Dithiothreitol; Escherichia coli; Escherichia coli Proteins; Lactose; Membrane Potentials; Membrane Transport Modulators; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Naphthoquinones; Plasmids; Proteolipids; Symporters

Plumbagin (2-methyl-5-hydroxy, 1:4 naphthoquinone) isolated from the roots of Plumbago zeylanica when administered to hyperlipidaemic rabbits, reduced serum cholesterol and LDL-Chol. by 53 to 86 percent and 61 to 91 percent respectively. It lowered cholesterol/phospholipid ratio by 45.8 percent and elevates the decreased HDL-Chol significantly. Further, Plumbagin treatment prevented the accumulation of cholesterol and triglycerides in liver and aorta and regressed atheromatous plaques of thoracic and abdominal aorta. Plumbagin treated hyperlipidaemic subjects excreted more fecal cholesterol and phospholipids. In conclusion-Plumbagin feeding brings about a definite regression of atheroma and prevents the accumulation of cholesterol and triglycerides in liver and aorta.

Topics: Animals; Aorta; Arteriosclerosis; Cholesterol; Cholesterol, HDL; Diet, Atherogenic; Feces; Hypolipidemic Agents; India; Liver; Naphthoquinones; Phospholipids; Plants, Medicinal; Rabbits; Triglycerides

We have previously reported that several naphthoquinones stimulated tyrosine-specific protein phosphorylation in isolated rat liver membranes. Our more recent study demonstrated a similar effect by orthovanadate, which concomitantly stimulated phosphorylation of protein-tyrosine and phosphatidylinositol (Ptd-Ins). Results presented here show a simultaneous increase in PtdIns phosphorylation along with stimulation of tyrosine-protein phosphorylation by naphthoquinones. This PtdIns kinase resembles the type I PtdIns kinase in that it was insensitive to adenosine inhibition. The product, nevertheless, comigrated with a PtdIns-4-phosphate standard in TLC using three different solvent systems. Stimulation of PtdIns phosphorylation by vanadate or naphthoquinones could be achieved in the following preparations: intact rat liver membranes, Triton X-100-solubilized membranes, solubilized membranes partially purified by Sephacryl chromatography, solubilized membranes purified by wheat germ agglutinin chromatography. The naphthoquinone or vanadate-activated PtdIns kinase activity could be isolated by antiphosphotyrosine antibody-agarose affinity chromatography. The relative potencies of a series of ring-substituted naphthoquinones in the stimulation of tyrosine-protein phosphorylation, PtdIns kinase activity, dithiothreitol-dependent oxygen consumption, and cytochrome c reduction were highly correlated. We conclude that oxidant(s) produced by redox cycling of naphthoquinones stimulated an adenosine-insensitive PtdIns kinase through tyrosine phosphorylation of the enzyme.

Topics: 1-Phosphatidylinositol 4-Kinase; Animals; Cell Membrane; Chromatography, Affinity; Kinetics; Liver; Male; Naphthoquinones; Phosphorylation; Phosphotransferases; Phosphotyrosine; Protein-Tyrosine Kinases; Rats; Rats, Inbred Strains; Tyrosine; Vanadates

Topics: Aerobiosis; Anaerobiosis; Escherichia coli; Free Radicals; Genotype; Mutagenicity Tests; Mutagens; Mutation; Naphthoquinones; Paraquat; Superoxide Dismutase; Superoxides

Plumbagin, a compound derived from the roots of Plumbago zeylanica (Chitramool) was studied for its effect on the development of antibiotic resistance using antibiotic sensitive strains of Escherichia coli and Staphylococcus aureus. A delayed growth was seen when these organisms were inoculated into the antibiotic (streptomycin/rifampicin) medium, due to development of resistance in some of the cells. However, the growth was completely prevented when the bacteria were grown in the medium containing antibiotic and plumbagin together, and this was attributed to prevention of development of antibiotic resistant cells.

Topics: Anti-Bacterial Agents; Culture Media; Drug Resistance, Microbial; Escherichia coli; Naphthoquinones; Plant Extracts; Staphylococcus aureus

The role of thiol groups in H+-gradient-dependent dipeptide transport in rabbit renal brush-border membrane vesicles was investigated using glycylsarcosine as the substrate. Treatment of the membrane vesicles with a thiol-group-reducing agent, dimercaptopropanol, stimulated Gly-Sar transport. On the other hand, treatment with thiol group oxidants such as 5,5'-dithiobis(2-nitrobenzoic acid), plumbagin and phenazine methosulfate inhibited Gly-Sar transport. These effects were irreversible, because washing the membranes after treatment failed to reverse the effects. Incubation of the membrane vesicles with phenylarsine oxide, a reagent which interacts specifically with vicinal dithiols, significantly inhibited Gly-Sar transport. In all cases, the stimulation or the inhibition of the dipeptide transport was primarily due to changes in the maximal velocity of the transport system, the apparent affinity constant remaining unaltered. These results demonstrate the involvement of one or more vicinal dithiol groups in the function of the renal dipeptide transport system and that these thiol groups must exist in reduced form to maintain maximal transport activity. In addition, these data indirectly suggest that a dithiol-disulfide interchange may play a role in the function of the renal dipeptide transport system.

Topics: Animals; Arsenicals; Biological Transport; Carrier Proteins; Dimercaprol; Dipeptides; Dithionitrobenzoic Acid; Kidney Cortex; Kinetics; Methylphenazonium Methosulfate; Microvilli; Naphthoquinones; Oxidation-Reduction; Protons; Rabbits; Structure-Activity Relationship; Sulfhydryl Compounds

Exposure of the plasmid pBR 322 to the aerobic xanthine oxidase reaction introduced single strand scissions and endonuclease III-sensitive sites. The latter may be residues of thymine glycol. Both forms of DNA damage were completely prevented by superoxide dismutase or catalase, whereas bovine serum albumin was much less effective. Mannitol and benzoate, added as scavengers of HO., and desferrioxamine or diethylene triamine pentaacetate, added to sequester Fe(III), also protected. These results indicate a metal-catalyzed interaction of O2- with H2O2, which produces HO. which, in turn, causes DNA strand scission and oxidation of thymine residues to thymine glycol. Plasmid isolated from aerobically-incubated cells contained more strand scissions and endonuclease III-sensitive sites than did plasmid from anaerobically-incubated cells, and a low molecular weight scavenger of O2- prevented the damage seen with the aerobic cells. Genetic defects in AP endonucleases rendered E. coli more susceptible to the dioxygen-dependent lethality of plumbagin, which mediates O2- production. Similarly, plasmid DNA, within the endonuclease-deficient cells, exhibited more strand scissions and endonuclease III-sensitive sites upon aerobic exposure to plumbagin than did endonuclease-sufficient cells, and a low molecular weight scavenger of O2- was protective. These results are consistent with the conclusions that strand scissions and formation of endonuclease III-sensitive sites are among the consequences of exposure of DNA to O2- plus H2O2, both in vitro and in vivo.

Topics: Catalase; Deoxyribonuclease (Pyrimidine Dimer); DNA Damage; DNA Repair; DNA, Bacterial; Endodeoxyribonucleases; Escherichia coli; Escherichia coli Proteins; Free Radicals; Hydrogen Peroxide; Hydroxides; Hydroxyl Radical; Manganese; Naphthoquinones; Oxygen; Plasmids; Superoxide Dismutase; Superoxides

Different conditions of oxidative stress were used to study their effects on membrane transport in Escherichia coli K-12. The oxidizing conditions included H2O2, plumbagin (a redox cycling compound that generates superoxide radicals [O2-]), and increased partial pressure of oxygen. Both superoxide radical-generating conditions and H2O2 treatments were found to cause a rapid decrease in proton motive force-dependent and -independent transport. H2O2-pretreated cells had the ability to rapidly recover both proton motive force-dependent and -independent transport. The induction required transcription and translation and was dependent on oxyR+ and katG+, providing evidence that these genes play crucial roles in the rapid recovery of transport. The effects of oxidatively induced loss of proton motive force on cell growth and macromolecular synthesis were also investigated.

Topics: Biological Transport, Active; Catalase; Cell Membrane; Chloramphenicol; Escherichia coli; Genes, Bacterial; Genes, Regulator; Hydrogen Peroxide; Mutation; Naphthoquinones; Oxidation-Reduction; Oxygen; Protein Biosynthesis; Protons; Rifampin; Transcription, Genetic

Mu dX phage was used to isolate three gene fusions to the lacZ gene (soi::lacZ; soi for superoxide radical inducible) that were induced by treatment with superoxide radical anion generators such as paraquat and plumbagin. The induction of beta-galactosidase in these fusion strains with the superoxide radical generating agents required aerobic metabolism. Hyperoxygenation (i.e., bubbling of cultures with oxygen gas) also induced the fusions. On the other hand, hydrogen peroxide did not induce the fusions at concentrations that are known to invoke an adaptive response. Introduction of oxyR, htpR, or recA mutations did not affect the induction. Two of the fusion strains exhibited increased sensitivity to paraquat but not to hydrogen peroxide. The third fusion strain showed no increased sensitivity to either agent. All three fusions were located in the 45- to 61-min region of the Escherichia coli chromosome.

Topics: Aerobiosis; Bacteriophage mu; beta-Galactosidase; DNA Repair; DNA Transposable Elements; Escherichia coli; Free Radicals; Genes; Genes, Bacterial; Hydrogen Peroxide; Naphthoquinones; Oxidation-Reduction; Oxygen; Paraquat; Recombinant Fusion Proteins; Recombinant Proteins; Recombination, Genetic; Superoxides

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

An activity stain has been devised for the dihydroxy-acid dehydratase. When applied to polyacrylamide gel electropherograms of crude soluble extracts of Escherichia coli, it detected a single electromorph. The intensity of staining increased with the amount of extract protein applied to the gel. Activity staining demonstrated that (a) anaerobically grown cells contain more extractable dehydratase activity than do aerobically grown cells; (b) exposure of E. coli to 4.2 atm O2 caused virtually complete loss of activity; (c) exposure of cells to paraquat or plumbagin in the presence of dioxygen, but not in its absence, caused a massive loss of activity. These data illustrate the utility of this activity stain and demonstrate that the dehydratase is inactivated by O2- generated within cells.

Topics: Escherichia coli; Hydro-Lyases; Naphthoquinones; Paraquat; Staining and Labeling; Superoxides

Increasing the intracellular flux of O-2 by incubating aerobic Escherichia coli with paraquat or plumbagin markedly lowered the alpha, beta-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells. This effect was not seen in the absence of dioxygen and was exacerbated by inhibiting protein biosynthesis with chloramphenicol. These effects of paraquat and of plumbagin were both time- and concentration-dependent. Transfer of E. coli from aerobic to anaerobic conditions caused a rebound of the dehydratase activity, in the continued presence of paraquat and of chloramphenicol, indicating the presence of a mechanism for reactivating this enzyme. The instability of the dehydratase activity in cell extracts was exacerbated by selective removal of superoxide dismutase, but not of catalase, by immunoprecipitation. Addition of exogenous superoxide dismutase reversed the effect of immunoprecipitation; whereas catalase or inactive superoxide dismutase were ineffective. We conclude that the dehydratase is inactivated by O-2. This could account for the bacteriostatic effects of dioxygen and of paraquat.

Topics: Catalase; Chloramphenicol; Escherichia coli; Hydro-Lyases; Naphthoquinones; Paraquat; Superoxide Dismutase; Superoxides

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: Drug Resistance, Microbial; Enzyme Inhibitors; Escherichia coli; Naphthoquinones; Penicillin Resistance; Penicillinase; Plant Extracts; Staphylococcus aureus

Among aerotolerant cells, Neisseria gonorrhoeae is very unusual because despite its obligately aerobic lifestyle and frequent isolation from purulent exudates containing polymorphonuclear leukocytes vigorously evolving O2- and H2O2, it contains no superoxide dismutase (SOD). Strains (14) of N. gonorrhoeae were compared with each other and with strains of Neisseria meningitidis, Neisseria mucosa, and Neisseria subflava under identical growth conditions for their contents of the oxy-protective enzymes catalase, peroxidase, and SOD, as well as respiratory chain proteins and activity. The absence of SOD from N. gonorrhoeae strains was demonstrated under a variety of oxygen-stress conditions. The neisserial species showed very different SOD, catalase, and peroxidase profiles. These profiles correlated well with the tolerance of the species to various intra- and extracellular oxygen insults. The high tolerance of N. gonorrhoeae for extracellular O2- and H2O2 appeared to be due to very high constitutive levels of peroxidase and catalase activity combined with a cell envelope impervious to O2-. Nevertheless, N. gonorrhoeae 19424 was much more sensitive to an intracellular flux of O2- than were the other (SOD-containing) neisserial species. The responses of N. gonorrhoeae and N. meningitidis respiratory and oxy-protective enzymes to growth under high and low oxygen tensions were followed, and a novel response, the apparent repression of the respiratory chain intermediates, respiration, and SOD, peroxidase, and catalase activity, was observed. The gonococcal catalase was partially purified and characterized. The results suggest that the very active terminal oxidase, low pO2 natural habitat, O2-stable catalase, and possibly the high glutathione content of the organism explain its aerobic survival in the absence of SOD.

Topics: Catalase; Free Radicals; Glutathione; Hydrogen Peroxide; Naphthoquinones; Neisseria; Oxygen; Superoxide Dismutase

Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.

Topics: Bacteriophage lambda; beta-Galactosidase; DNA Repair; Escherichia coli; Hydrogen Peroxide; Light; Lysogeny; Mutagenicity Tests; Naphthoquinones; Operon; Riboflavin

Topics: Animals; Antiprotozoal Agents; Chlorpromazine; Clofazimine; Electron Transport; Leishmania donovani; Leishmania mexicana; Leishmaniasis; Leishmaniasis, Visceral; Methylene Blue; Methylphenazonium Methosulfate; Mice; Mice, Inbred BALB C; Naphthoquinones; Nifurtimox; Vitamin K

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

Topics: Animals; Cholesterol; Dogs; Epididymis; Hydrolases; Male; N-Acetylneuraminic Acid; Naphthoquinones; Organ Size; Proteins; Reproduction; RNA; Sialic Acids; Testis; Vas Deferens

The effects of chemical modification of the lactose carrier of Escherichia coli on galactoside binding (in overproducing strains) and on transport was examined. Both the modifying reagents diethylpyrocarbonate and rose bengal and the thiol reagents phenylarsinoxide and plumbagin can completely inhibit the binding of the substrate p-nitrophenyl alpha-D-galactopyranoside to the carrier. If care is taken to inhibit galactoside binding only partially, the loss of transport is found to parallel the loss of binding sites. The modified carrier molecules are completely inactive, while the remaining active carrier molecules evince normal transport and binding parameters. The binding of galactoside protects the carrier partially against these forms of chemical modification. In view of these observations, the results of previous chemical modification studies [Padan, E., Patel, L. and Kaback, H.R. (1979) Proc. Natl Acad. Sci. USA, 76, 6221-6225; Konings, W.N. and Robillard, G.T. (1982) Proc. Natl Acad. Sci. USA, 79, 5480-5484] must be re-interpreted. Our results stress the utility of studying substrate binding, the first step in the transport cycle.

Topics: Arsenicals; Binding Sites; Biological Transport; Chemical Phenomena; Chemistry; Diethyl Pyrocarbonate; Escherichia coli; Escherichia coli Proteins; Formates; Galactosides; Glycosides; Membrane Transport Proteins; Monosaccharide Transport Proteins; Naphthoquinones; Protein Binding; Symporters

Streptococcus sanguis, whose growth appears to be independent of the availability of iron, makes no hemes, contains neither catalase nor peroxidase, and can accumulate millimolar concentration levels of H2O2 during aerobic growth. It possesses a single manganese-containing superoxide dismutase whose concentration can be varied over a 50-100-fold range by manipulating the availability of oxygen during growth. Cell extracts contain a soluble NADH-plumbagin diaphorase which mediates O2- production in vitro and presumably also in vivo. Plumbagin increased oxygen consumption by S. sanguis and imposed an oxygen-dependent toxicity. Cells grown aerobically and containing elevated levels of superoxide dismutase were resistant to this toxicity. Dimethyl sulfoxide, which was shown to permeate S. sanguis freely, was used as an indicating scavenger of OH. An in vitro enzymic source of O2- plus H2O2 generated formaldehyde from dimethyl sulfoxide, an indication of OH. production. Either superoxide dismutase or catalase inhibited this OH. production and iron salts augmented it. Intact, aerobic cells of S. sanguis also gave evidence of OH. production, in the presence of plumbagin, but all of it appeared to be generated outside the cells. In addition, 0.5 M dimethyl sulfoxide did not diminish the oxygen-dependent toxicity of plumbagin. We conclude that, in S. sanguis, O2- can exert a toxic effect independent of the production of OH..

Topics: Dimethyl Sulfoxide; Enzyme Induction; Free Radicals; Hydroxides; Hydroxyl Radical; Iron; Kinetics; Naphthoquinones; Oxygen; Streptococcus; Superoxide Dismutase; Superoxides

The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers. A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity. Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition. These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide. Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline. The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate. The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+). These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.

Topics: Arsenicals; Biological Transport, Active; Dithiothreitol; Escherichia coli; Escherichia coli Proteins; Kinetics; Lactose; Membrane Transport Proteins; Monosaccharide Transport Proteins; Naphthoquinones; Oxidation-Reduction; Proline; Sulfhydryl Reagents; Symporters

Topics: Alkaloids; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Guinea Pigs; Naphthoquinones; Spectrophotometry

Topics: Animals; Contraceptive Agents; Female; Male; Naphthoquinones; Plant Extracts; Plants, Medicinal; Rats

Lactobacillus plantarum is aerotolerant during log-phase growth on glucose, but is an obligate aerobe on polyols. Respiration was cyanide resistant and under certain conditions was associated with the accumulation of millimolar concentrations of H(2)O(2). On glucose, optimal growth was observed in the absence of O(2). Extracts of L. plantarum did not catalyze the reduction of paraquat by reduced nicotinamide adenine dinucleotide, but plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) was readily reduced. Such extracts produced O(2) (-) in the presence of NADH plus plumbagin. Plumbagin caused a 10-fold increase in the rate of respiration of intact cells in the presence of glucose and also imposed a loss of viability which was dependent upon both glucose and O(2). Although extracts of L. plantarum were devoid of true superoxide dismutase activity, this organism was comparable to superoxide dismutase-containing species in its resistance toward hyperbaric O(2) and toward the oxygen-dependent lethality of plumbagin. L. plantarum required Mn-rich media and actively accumulated Mn(II). Soluble extracts were found to contain approximately 9 mug of Mn per mg of protein and 75 to 90% of this Mn was dialyzable. Such extracts exhibited a dialyzable and ethylenediaminetetraacetic acid-inhibitable ability to scavenge O(2) (-). This O(2) (-)-scavenging activity was due to the dialyzable Mn(II) present in these extracts and could be mimicked by MnCl(2). Cells grown in Mn-rich media were enriched in dialyzable Mn and were more resistant toward oxygen toxicity and toward the oxygen-dependent plumbagin toxicity than were cells grown in Mn-deficient media. L. plantarum exhibited no nutritional requirement for iron and little or no iron was present in these cells, even when they were grown in iron-rich media. L. plantarum thus appears to use millimolar levels of Mn(II) to scavenge O(2) (-), much as most other organisms use micromolar levels of superoxide dismutases.

Topics: Hydrogen Peroxide; Lactobacillus; Manganese; Naphthoquinones; Oxygen; Oxygen Consumption; Superoxide Dismutase; Superoxides

A previous study of the aerotolerant bacterium Lactobacillus plantarum, which lacks superoxide dismutase (SOD), demonstrated that it possesses a novel substitute for this defensive enzyme. Thus, L. plantarum contains 20 to 25 mM Mn(II),m in a dialyzable form, which is able to scavenge O2- apparently as effectively as do the micromolar levels of SOD present in most other organisms. This report describes a survey of the lactic acid bacteria. The substitution of millimolar levels of Mn(II) for micromolar levels of SOD is a common occurrence in this group of microorganisms, which contained either SOD or high levels of Mn(II), but not both. Two strains were found which had neither high levels of Mn(II) nor SOD, and they were, as was expected, very oxygen intolerant. Lactic acid bacteria containing SOD grew better aerobically than anaerobically, whereas the organisms containing Mn(II) in place of SOD showed aerobic growth which was best, at best, equal to anaerobic growth. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) increases the rate of O2- production in these organisms. Lactobacillus strains containing high intracellular Mn(II) were more resistant to the oxygen-dependent toxicity of plumbagin than were strains containing lower levels of Mn(II). The results support the conclusion that a high internal level of Mn(II) provides these organisms with an important defence against endogenous O2-.

Topics: Aerobiosis; Anaerobiosis; Escherichia coli; Lactobacillus; Manganese; Naphthoquinones; Oxygen; Streptococcaceae; Superoxide Dismutase; Superoxides

Topics: Animals; Cell Division; Cells, Cultured; Chick Embryo; Fibroblasts; Heart; Mitosis; Naphthoquinones

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Antineoplastic Agents, Phytogenic; Bacteria; Drug Evaluation, Preclinical; Female; Fibrosarcoma; Fungi; Leukemia, Experimental; Male; Mice; Naphthoquinones; Neoplasms, Experimental

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Blood Coagulation; Liver; Male; Naphthoquinones; Rats