naphthoquinones and shikonin

Shikonin, the primary active compound found in the rhizome of the traditional Chinese medicinal herb known as "ZiCao", exhibits a diverse range of pharmacological effects. This drug has a wide range of uses, including as an anti-inflammatory, antioxidant, and anti-cancer agent. It is also effective in promoting wound healing and treating autoimmune diseases such as multiple sclerosis, diabetes, asthma, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, and rheumatoid arthritis. Although shikonin has a wide range of applications, its mechanisms are still not fully understood. This review article provides a comprehensive overview of the recent advancements in the use of shikonin for the treatment of immune-related diseases. The article also delves into the anti-inflammatory and immunoregulatory mechanisms of shikonin and offers insights into the inflammation and immunopathogenesis of related diseases. Overall, this article serves as a valuable resource for researchers and clinicians working in this field. These findings not only provide significant new information on the effects and mechanisms of shikonin but also establish a foundation for the development of clinical applications in treating autoimmune diseases.

Topics: Anti-Inflammatory Agents; Autoimmune Diseases; Humans; Inflammation; Naphthoquinones

Shikonin is one of the major phytochemical components of Lithospermum erythrorhizon (Purple Cromwell), which is a type of medicinal herb broadly utilized in traditional Chinese medicine. It is well established that shikonin possesses remarkable therapeutic actions on various diseases, with the underlying mechanisms, pharmacokinetics and toxicological effects elusive. Also, the clinical trial and pharmaceutical study of shikonin remain to be comprehensively delineated.. The present review aimed to systematically summarize the updated knowledge regarding the therapeutic actions, pharmacokinetics, toxicological effects, clinical trial and pharmaceutical study of shikonin.. The information contained in this review article were retrieved from some authoritative databases including Web of Science, PubMed, Google scholar, Chinese National Knowledge Infrastructure (CNKI), Wanfang Database and so on, till August 2021.. Shikonin exerts multiple therapeutic efficacies, such as anti-inflammation, anti-cancer, cardiovascular protection, anti-microbiomes, analgesia, anti-obesity, brain protection, and so on, mainly by regulating the NF-κB, PI3K/Akt/MAPKs, Akt/mTOR, TGF-β, GSK3β, TLR4/Akt signaling pathways, NLRP3 inflammasome, reactive oxygen stress, Bax/Bcl-2, etc. In terms of pharmacokinetics, shikonin has an unfavorable oral bioavailability, 64.6% of the binding rate of plasma protein, and enhances some metabolic enzymes, particularly including cytochrome P450. In regard to the toxicological effects, shikonin may potentially cause nephrotoxicity and skin allergy. The above pharmacodynamics and pharmacokinetics of shikonin have been validated by few clinical trials. In addition, pharmaceutical innovation of shikonin with novel drug delivery system such as nanoparticles, liposomes, microemulsions, nanogel, cyclodextrin complexes, micelles and polymers are beneficial to the development of shikonin-based drugs.. Shikonin is a promising phytochemical for drug candidates. Extensive and intensive explorations on shikonin are warranted to expedite the utilization of shikonin-based drugs in the clinical setting.

Topics: Naphthoquinones; NF-kappa B; Pharmaceutical Preparations; Phosphatidylinositol 3-Kinases

Shikonin and its derivatives are excellent representatives of biologically active naphthoquinones. A wide range of investigations carried out in the last few decades validated their pharmacological efficacy. Besides having magnificent therapeutic potential, shikonin and its derivatives suffer from various pharmacokinetic, toxicity, and stability issues like poor bioavailability, nephrotoxicity, photodegradation, etc. Recently, various research groups have developed an extensive range of formulations to tackle these issues to ease their path to clinical practice. The latest formulation approaches have been focused on exploiting the unique features of novel functional excipients, which in turn escalate the therapeutic effect of shikonin. Moreover, the codelivery approach in various drug delivery systems has been taken into consideration in a recent while to reduce toxicity associated with shikonin and its derivatives. This review sheds light on the essential reports and patents published related to the array of formulations containing shikonin and its derivatives.

Topics: Drug Delivery Systems; Naphthoquinones

Shikonin and its enantiomeric analogue, alkaninn, are prevailing natural lead compounds in the drug discovery and development of anticancer agents. Despite having numerous biological effects, the most important activity reported for shikonin derivatives is the antitumor effect which is exerted through various mechanisms such as induction of apoptosis and autophagy. The design, synthesis, and development of new shikonin derivatives are continuously performed with the aim of promoting therapeutic effects through increasing cytotoxicity against cancer cells and simultaneously reducing toxicity on normal cells. In spite of significant advances in the development of shikonin derivatives in recent years and the publication of some reviews in this regard, the structural classification, synthesis methods, as well as the diversity of the anti-tumor mechanism of action of these compounds have not been well considered. This review aims to provide comprehensive data in this regard by reviewing studies conducted over the last two decades (from 2000 until now).

Topics: Antineoplastic Agents; Apoptosis; Humans; Naphthoquinones; Neoplasms

Shikonin is one of the primary active components extracted from the dried root of

Topics: Humans; Inflammation; Lithospermum; Naphthoquinones; Skin Diseases

Coronaviruses represent global health threat. In this century, they have already caused two epidemics and one serious pandemic. Although, at present, there are no approved drugs and therapies for the treatment and prevention of human coronaviruses, several agents, FDA-approved, and preclinical, have shown in vitro and/or in vivo antiviral activity. An in-depth analysis of the current situation leads to the identification of several potential drugs that could have an impact on the fight against coronaviruses infections. In this review, we discuss the virology of human coronaviruses highlighting the main biological targets and summarize the current state-of-the-art of possible therapeutic options to inhibit coronaviruses infections. We mostly focus on FDA-approved and preclinical drugs targeting viral conserved elements.

Topics: Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antiviral Agents; Azoles; Coronavirus; Coronavirus Infections; COVID-19; COVID-19 Drug Treatment; Dipeptidyl Peptidase 4; Enzyme Inhibitors; Humans; Isoindoles; Naphthoquinones; Organoselenium Compounds; Severe Acute Respiratory Syndrome

Pyruvate kinase M2 is a critical enzyme that regulates cell metabolism and growth under different physiological conditions. In its metabolic role, pyruvate kinase M2 catalyzes the last glycolytic step which converts phosphoenolpyruvate to pyruvate with the generation of ATP. Beyond this metabolic role in glycolysis, PKM2 regulates gene expression in the nucleus, phosphorylates several essential proteins that regulate major cell signaling pathways, and contribute to the redox homeostasis of cancer cells. The expression of PKM2 has been demonstrated to be significantly elevated in several types of cancer, and the overall inflammatory response. The unusual pattern of PKM2 expression inspired scientists to investigate the unrevealed functions of PKM2 and the therapeutic potential of targeting PKM2 in cancer and other disorders. Therefore, the purpose of this review is to discuss the mechanistic and therapeutic potential of targeting PKM2 with the focus on cancer metabolism, redox homeostasis, inflammation, and metabolic disorders. This review highlights and provides insight into the metabolic and non-metabolic functions of PKM2 and its relevant association with health and disease.

Topics: Adenosine Triphosphate; Atherosclerosis; Carrier Proteins; Cell Proliferation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glycolysis; Homeostasis; Humans; Inflammation; Inflammatory Bowel Diseases; Insulin; Kidney Diseases; Liver; Membrane Proteins; Metabolic Diseases; Naphthoquinones; Neoplasm Metastasis; Neoplasms; Neuralgia; Oxidants; Oxidation-Reduction; Protein Isoforms; Sepsis; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Tissue Distribution

Shikonin is the major bioactive component extracted from the roots of Lithospermum erythrorhizon which is also known as "Zicao" in Traditional Chinese Medicine (TCM). Recent studies have shown that shikonin demonstrates various bioactivities related to the treatment of cancer, inflammation, and wound healing. This review aimed to provide an updated summary of recent studies on shikonin. Firstly, many studies have demonstrated that shikonin exerts strong anticancer effects on various types of cancer by inhibiting cell proliferation and migration, inducing apoptosis, autophagy, and necroptosis. Shikonin also triggers Reactive Oxygen Species (ROS) generation, suppressing exosome release, and activate anti-tumor immunity in multiple molecular mechanisms. Examples of these effects include modulating the PI3K/AKT/mTOR and MAPKs signaling; inhibiting the activation of TrxR1, PKM2, RIP1/3, Src, and FAK; and regulating the expression of ERP57, MMPs, ATF2, C-MYC, miR-128, and GRP78 (Bip). Next, the anti-inflammatory and wound-healing properties of shikonin were also reviewed. Furthermore, several studies focusing on shikonin derivatives were reviewed, and these showed that, with modification to the naphthazarin ring or side chain, some shikonin derivatives display stronger anticancer activity and lower toxicity than shikonin itself. Our findings suggest that shikonin and its derivatives could serve as potential novel drug for the treatment of cancer and inflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Endoplasmic Reticulum Chaperone BiP; Humans; Lithospermum; Naphthoquinones; Neoplasms; Wound Healing

Shikonin is a natural compound isolated from herbs and traditional medicines that have been used in a number of countries to treat various illnesses including inflammation, virus infection and cancer for centuries. Recent studies have shed light on the molecular mechanisms underlying these biological activities of Shikonin. Here we review the latest advances in our understanding of this compound class in the anti-cancer regimen. We focus on signaling pathways and cellular targets involved in the anticancer activity of Shikonin. We also briefly discuss approaches in evaluating the in vivo bioactivity and drug delivery of Shikonin in the anti-cancer treatment. Subsequently, we highlight recently developed strategies in the chemical and biogenic synthesis of Shikonin and summarize the structure-activity relationship studies of Shikonin. We anticipate that these lines of information would facilitate the functional identification and future clinical development of Shikonin and its derivatives in the combat against cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Drug Delivery Systems; Humans; Lithospermum; Naphthoquinones; Plant Roots; Signal Transduction

Phytochemicals gained considerable interest during the past years as source to develop new treatment options for chemoprevention and cancer therapy. Motivated by the fact that a majority of established anticancer drugs are derived in one way or another from natural resources, we focused on shikonin, a naphthoquinone with high potentials to be further developed as preventive or therapeutic drug to fight cancer. Shikonin is the major chemical component of Lithospermum erythrorhizon (Purple Cromwell) roots. Traditionally, the root extract has been applied to cure dermatitis, burns, and wounds. Over the past three decades, the anti-inflammatory and anticancer effects of root extracts, isolated shikonin as well as semi-synthetic and synthetic derivatives and nanoformulations have been described. In vitro and in vivo experiments were conducted to understand the effect of shikonin at cellular and molecular levels. Preliminary clinical trials indicate the potential of shikonin for translation into clinical oncology. Shikonin exerts additive and synergistic interactions in combination with established chemotherapeutics, immunotherapeutic approaches, radiotherapy and other treatment modalities, which further underscores the potential of this phytochemical to be integrated into standard treatment regimens.

Topics: Animals; Antineoplastic Agents, Phytogenic; Drugs, Chinese Herbal; Humans; Models, Molecular; Naphthoquinones; Neoplasms; Structure-Activity Relationship

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.

Topics: Artemisinins; Axenic Culture; Berberine; Biological Products; Biotechnology; Cell Culture Techniques; Metabolic Engineering; Naphthoquinones; Paclitaxel; Phytochemicals; Plant Cells; Plants; Secondary Metabolism; Tissue Culture Techniques

Shikonin, alkannin and their derivatives, the main ingredient of Lithospermum erythrorhizon and Arnebia euchroma (Royle) Johnst native to Inner Mongolian and Northwest of China respectively, hold promising potentials for antitumor effects via multiple-target mechanisms. This review will emphasize the importance of their antitumor activity in apoptosis, necroptosis and immunogenic cell death, and expound the relationship of their antitumor activity and naphthoquinone scaffold that could generate ROS and alkylating agent. Meanwhile, the antitumor mechanisms of naturally-occurring shikonin, alkannin and their derivatives, which were divided into the direct interaction involved in alkylating agent, covalently binding the DNA and protein, as well as the indirect interaction mediated by ROS, nonspecifically influencing the mitochondria or multiple signal pathways, will be systematically summarized and discussed.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Death; Drug Screening Assays, Antitumor; Humans; Lithospermum; Naphthoquinones; Neoplasms; Reactive Oxygen Species

Shikonins are commercially important secondary compounds, known for array of biological activities such as antimicrobial, insecticidal, antitumor, antioxidants, etc. These compounds are usually colored and therefore have application in food, textiles and cosmetics. Shikonin and its derivatives, which are commercially most important of the naphthoquinone pigments, are distributed among members of the family Boraginaceae. These include different species of Lithospermum, Arnebia, Alkanna, Anchusa, Echium and Onosma. The growing demand for plant-based natural products has made this group of compounds one of the enthralling targets for their in vitro production. The aim of this review is to highlight the recent progress in production of shikonins by various biotechnological means. Different methods of increasing the levels of shikonins in plant cells such as selection of cell lines, optimization of culture conditions, elicitation, in situ product removal, genetic transformation and metabolic engineering are discussed. The experience of different researchers working worldwide on this aspect is also considered. Further, to meet market demand, the needs for continuous and reliable production systems, as well as future prospects, are included.

Topics: Bioengineering; Boraginaceae; Naphthoquinones; Plant Extracts; Tissue Culture Techniques

Shikonin is the major constituent of the root of Lithospermum erythrorhizon, which has been used in traditional Chinese medicine to treat external wounds, burns, or dermatitis for centuries. Nowadays, this root is commonly used as an herbal medicine against cancer. Studies carried out over the past 30 years have demonstrated that many of the effects historically associated with the use of this root have a scientific basis, with shikonin and its derivatives being responsible for its pharmacological properties. These include both anti-inflammatory and anticancer effects. While previous summaries have focused on the pharmacokinetics and toxicity of shikonin, the aim of this review is to report on the most current findings with regard to shikonin's antitumor activity by summarizing and comparing the various studies published in the last ten years and discussing the pharmacological aspects that make shikonin a promising anticancer agent.

Topics: Anti-Inflammatory Agents; Antineoplastic Agents; Humans; Medicine, Chinese Traditional; Naphthoquinones; Neoplasms; Plant Roots

The naphthoquinone shikonin is the main active principle of Zicao, a traditional Chinese herbal medicine made from the dried root of Lithospermum erythrorhizon. Studies carried out over the past 30 years have provided a scientific basis for the use of Zicao which has been long employed in folk medicine to treat a variety of inflammatory and infectious diseases. In particular, shikonin has been shown to possess many diverse properties, including antioxidant, anti-inflammatory, antithrombotic, antimicrobial, and wound healing effects. The fact that shikonin shows so many beneficial properties has increased the interest in this molecule dramatically, especially in the past few years. The aim of this review is to provide an update of the new data published on shikonin, whose wide spectrum of pharmacological effects as well as pharmacokinetic properties and toxicity make it a highly interesting target molecule.

Topics: Anti-Infective Agents; Anti-Inflammatory Agents; Antioxidants; Fibrinolytic Agents; Humans; Lithospermum; Medicine, Chinese Traditional; Naphthoquinones; Plant Roots; Plants, Medicinal; Wound Healing

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

Shikonin, the main active ingredient of Lithospermum, and its derivatives have been proved to have antitumor effects, and the anti-tumor mechanisms involve multiple targets. Based on recent literatures, this review focuses on the antitumor effects and its mechanisms. More emphases are given on the aspects of induction of apoptosis, induction of necrosis, acting on matrix metalloproteinase, acting on the protein tyrosine kinase and antiangiogenesis. The current status and problems of shikonin derivatives in antitumor effects are simply summarized and lookout for the development of antitumor drugs with shikonin as leading compounds.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Drugs, Chinese Herbal; Humans; Lithospermum; Matrix Metalloproteinase 9; Naphthoquinones; Necrosis; Neoplasms; Neovascularization, Pathologic; Plants, Medicinal; Protein-Tyrosine Kinases; Reactive Oxygen Species

Shikonin and its derivatives are the main components of red pigment extracts from Lithospermum erythrorhizon, whose medicinal properties have been confirmed for a long history, and have aroused great interest as the hallmark molecules responsible for their significant biological activities, especially for their striking anticancer effects.. Areas covered in this paper include a review of the total synthesis, biological effects and mechanisms of shikonin and its derivatives for their anticancer activities in the past decade, basing on literature and patents. The current state and problems are also discussed.. At present, screening for anticancer shikonin derivatives is based on cellular level to find compounds with stronger cytotoxicity. Though several compounds have been discovered with striking cytotoxicity in vitro, however, no selectivity was observed and undoubtedly, the further outcomes have been disappointing because of their great damage to normal cells. Meanwhile, the presumed mechanisms of action are also established in terms of their cytotoxicity. From a pharmacological point of view, most of the shikonin derivatives are at an early stage of their development, and thus it is difficult to determine the exact effectiveness in cancer treatment. With research in this field going deeper, it can be expected that, despite the difficulties, shikonin derivatives as potential anticancer agents will soon follow.

Topics: Animals; Antineoplastic Agents, Phytogenic; Drug Design; Humans; Lithospermum; Naphthoquinones; Neoplasms; Patents as Topic; Plant Extracts

The proteasome is a multicatalytic protease complex whose activity is required for the growth of normal or tumor cells. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that the proteasome could be a target of chemotherapy. Studies suggest that traditional Chinese medicine (TCM) is an effective approach for cancer treatment. Here we reviewed several TCMs for their potential in treatment of cancer. This short review focuses mainly on the TCMs that potentially target the tumor cellular proteasome and NF-kappaB pathway whose activation is dependent on the proteasome activity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Benzyl Compounds; Curcumin; Diterpenes; Drug Delivery Systems; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Epoxy Compounds; Humans; Medicine, Chinese Traditional; Molecular Structure; Naphthoquinones; Neoplasms; Pentacyclic Triterpenes; Phenanthrenes; Phenols; Proteasome Endopeptidase Complex; Signal Transduction; Triterpenes

Alkannins and Shikonins (A/S) are chiral-pairs of naturally occurring isohexenylnaphthazarins. They are found in the external layer of the roots of at least a hundred and fifty species that belong mainly to the genera Alkanna, Lithospermum, Echium, Onosma and Arnebia of the Boraginaceae family. Their occurrence in Jatropha glandulifera, a member of the Euphorbiaceae, should be considered as an exception. Pharmaceutical formulations with wound healing properties based on A/S have been in the market for many years. Although their wound-healing, anti-inflammatory, antimicrobial, antioxidant, antithrombotic and antitumor properties have been extensively documented, significant insight into their specific molecular pathways and mechanisms was hindered until recently. With the establishment of viable synthetic and biosynthetic routes of A/S and the synthesis of specific derivatives that were discovered the last few years, the effects of those compounds in the molecular-cell biology of human tissues in health and disease have just started being explored in depth, revealing a new class of drugs that hold promise as the basis for many valuable therapeutic targets. In the recent years, a wealth of new information arising from research efforts, on the wound healing properties of A/S has been accumulated. In this paper we review the findings and advances on the molecular and biological properties of A/S that promote wound healing.

Topics: Animals; Computer Simulation; Humans; Models, Chemical; Molecular Structure; Naphthoquinones; Stereoisomerism; Wound Healing

Literature data on the production of naphthoquinoine pigment, shikonin, by the intact plants and cell cultures derived from the members of Boraginaceae family have been reviewed. The results of our own studies on generation of highly productive Arnebia euchroma cell lines able to accumulate up to 20% of shikonin in dry biomass are presented. Data on localization, application of naphthoquinoine pigments, peculiarities and enzyme control of their biosynthesis and transportation within plant cells and in culture in vitro are summarized.

Topics: Boraginaceae; Models, Molecular; Molecular Conformation; Naphthoquinones; Pigments, Biological; Stereoisomerism

Secologanin, a secoiridoid glucoside, is a pivotal terpenoid intermediate in the biosynthesis of biologically active monoterpenoid indole alkaloids such as reserpine, ajmaline, and vinblastine which are biosynthesized via strictosidine, an alkaloidal glucoside, formed from secologanin and tryptamine. In secologanin biosynthesis, the oxidative cleavage process of loganin to secologanin and the hydroxylation of 7-deoxyloganin to loganin have remained unknown enzymologically and mechanistically. Cornoside is a unique glucoside with 4-hydroxy-2,5-cyclohexadien-1-one (benzoquinol) ring and is widespread in families such as Cornaceae, Oleaceae, and Scrophulariaceae but its biosynthesis, especially the oxidative process, remain to be investigated. Shikonin is a red naphthazarin pigment derived from the roots of Lithospermum erythorhizon and produced biotechnologically by cell cultures. Its biosynthesis including the production regulation mechanism has been investigated in detail. However, the naphthazarin ring formation process, probably starting with the hydroxylation of the side chain of geranylhydroquinone, a key intermediate at the late stage of shikonin biosynthesis, remained unknown. In the present review, cytochrome P450 monooxygenases involved in the biosyntheses of three structurally and biosynthetically interesting compounds, secologanin, cornoside, and shikonin, a described together with the results of previous and recent biosynthetic studies. The biosyntheses of related compounds are also discussed.

Topics: Cyclohexanones; Cytochrome P-450 Enzyme System; Glucosides; Indole Alkaloids; Iridoid Glucosides; Iridoids; Naphthoquinones; Oxidation-Reduction; Plants; Secologanin Tryptamine Alkaloids; Tryptamines; Vinca Alkaloids

The naphthoquinone pigment, shikonin, isolated from Lithospermum erythrorhizon Sieb. et Zucc.(Boraginaceae) and its derivatives are the active components isolated from the Chinese herbal therapeutic, Zicao. Historically, Zicao root extracts have been used to treat macular eruption, measles, sore-throat, carbuncles and burns. Multiple pharmacological actions have been attributed to shikonin, e.g. antiinflammatory, antigonadotropic and anti-HIV-1 activity. In this review, several therapeutic applications of shikonin will be summarized including its pleiotropic, antiinflammatory and antitumour effects. Widely diverse and sometimes conflicting activities have been attributed to shikonin, e.g. wound healing, enhanced granuloma formation, suppression of local acute inflammatory reactions, inhibition of angiogenesis, inhibition of select chemokine ligands, inhibition of DNA topoisomerase activity, inhibition of platelet activation and antimicrobial activity. Comparison of the various reported mechanisms of action for shikonin lead us to hypothesize that shikonin is an effective inhibitor of protein-protein interaction with multiple targets in both the intracellular and extracellular compartments. This general inhibitory effect can account for the broad spectrum of shikonin biological and pharmacological activities.

Topics: Animals; Anti-Infective Agents; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Granuloma; Humans; Inflammation; Lithospermum; Mast Cells; Naphthoquinones; Neoplasms; Neutrophils; Phytotherapy; Plant Extracts; Platelet Aggregation Inhibitors; Prostaglandin-Endoperoxide Synthases; Respiratory Burst; Signal Transduction; Wound Healing

Topics: Anti-Bacterial Agents; Antineoplastic Agents, Phytogenic; Culture Media; Culture Techniques; Medicine, Chinese Traditional; Naphthoquinones; Plants, Medicinal

Clinical morphological efficiency of local application of a new biopolymeric film was studied. The film was based on methylcellulose derivatives and contained shikonin (preparation of plant origin) and its esters isolated from Lithospermum erythrorhizon L. cell culture. Combined therapy of 30 patients (34-72 years) with erosive ulcerative lichen planus and leukoplakia of the buccal mucosa was carried out. Local application of the new drug led to more rapid pain relief, epithelialization of the inflammatory destructive foci in the buccal mucosa, and reduced the intensity of morphological signs of lesions in the studied patient population.

Topics: Adult; Aged; Anti-Inflammatory Agents, Non-Steroidal; Biopolymers; Drugs, Chinese Herbal; Female; Humans; Inflammation; Leukoplakia, Oral; Lichen Planus, Oral; Male; Methylcellulose; Middle Aged; Mouth Mucosa; Naphthoquinones; Oral Ulcer

The enantiomeric naphthoquinones alkannins and shikonins (A/S) have been established as potent wound healing agents. The purpose of this study was to evaluate the effectiveness of an A/S based ointment for humans on second intention wound healing in the dog, as compared to wound flushing with Lactated Ringer's solution (LRS). Ten mixed breed dogs, aged 2 to 5 y, were used. One 2.5 × 2.5 cm full-thickness skin defect was created on the lateral aspect of each arm for subjective evaluation, laser-Doppler flowmetry (LDF), and planimetry. Additionally, 3 matching 2 × 2 cm wounds were created on opposite sides of the dorsal midline for histologic evaluation. Wounds were treated once daily with the A/S based ointment on the right side and by flushing with LRS on the left until healed (about 20 d). During the healing process, tissue perfusion (mean LDF value) was found to be significantly higher on the side treated with the A/S based ointment compared with the LRS-treated side. Histologically, angiogenesis (on days 4 and 11), collagen production score (on days 4, 11, and 20), and epithelial thickness score (on day 11) were significantly higher in the wounds treated with the A/S based ointment. Wound size, as evaluated by planimetry, decreased significantly from day 0 to day 20 on both sides, but no significant differences were found between the A/S based ointment and LRS-treated wounds.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Dogs; Drug Administration Schedule; Drug Combinations; Female; Forelimb; Isotonic Solutions; Laser-Doppler Flowmetry; Male; Naphthoquinones; Ringer's Lactate; Skin; Soft Tissue Injuries; Therapeutic Irrigation; Treatment Outcome; Wound Healing

Recently, intelligent packaging has emerged for monitoring food quality in food industry. This study aimed to develop the electrospun HACC/PCL/SKN nanofibrous films with improved antimicrobial and antioxidant activity as intelligent packaging to monitor food freshness. The SKN loading resulted in nanoscale uniform fibers (approximately 55.0 nm), and the HACC/PCL/SKN nanofibrous films presented improved hydrophobicity, barrier properties and mechanical properties. Release kinetics study demonstrated that the loading effect led to a sustained release of SKN from fibers. The HACC/PCL film containing 2 wt% SKN showed good antibacterial effect during 24 h, suggesting enhanced antimicrobial activity. Moreover, the SKN-based solutions and films exhibited pH-responsive color changes from red (pH 2) to blue-purple (pH 12). Finally, the HACC/PCL/SKN film effectively provided a spoilage indication for shrimp stored at different temperatures for 3 days by color changes. This work provides a promising strategy for developing multi-functional film as an intelligent packaging in food industry.

Topics: Anthocyanins; Anti-Infective Agents; Chitosan; Food Packaging; Hydrogen-Ion Concentration; Nanofibers; Naphthoquinones; Polyesters

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Topics: Gene Expression Regulation, Plant; Lipids; Naphthoquinones; Plant Proteins

Oxaliplatin (OXA) has been recognized as a third-generation platinum-based chemotherapeutic agent with stellar therapeutic efficacy in managing colorectal cancer (CRC). Nevertheless, resistance to OXA in CRC patients hinders its effectiveness. Shikonin (SHI), a natural naphthoquinone derived from Arnebia euchroma (Royle) Johnst., features a broad pharmacological profile and minimal toxicities. To assess the synergism of SHI and OXA towards OXA-resistant CRC cells (HCT116

Topics: Animals; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Endoplasmic Reticulum Stress; Humans; Mice; Naphthoquinones; Oxaliplatin; Reactive Oxygen Species

Shikonin is a red naphthoquinone natural product from plants with high economical and medical values. The para-hydroxybenzoic acid geranyltransferase (PGT) catalyzes the key regulatory step of shikonin biosynthesis. PGTs from Lithospermum erythrorhizon have been well-characterized and used in industrial shikonin production. However, its perennial medicinal plant Arnebia euchroma accumulates much more pigment and the underlying mechanism remains obscure. Here, we discovered and characterized the different isoforms of AePGTs. Phylogenetic study and structure modeling suggested that the N-terminal of AePGT6 contributed to its highest activity among 7 AePGTs. Indeed, AePGT2 and AePGT3 fused with 60 amino acids from the N-terminal of AePGT6 showed even higher activity than AePGT6, while native AePGT2 and AePGT3 don't have catalytic activity. Our result not only provided a mechanistic explanation of high shikonin contents in Arnebia euchroma but also engineered a best-performing PGT to achieve the highest-to-date production of 3-geranyl-4-hydroxybenzoate acid, an intermedium of shikonin.

Topics: Boraginaceae; Geranyltranstransferase; Naphthoquinones; Phylogeny

In the recent past, the occurrence of fungal infections has increased drastically and candidiasis, caused prominently by Candida albicans, is foremost among them which has caused significant mortality and morbidity majorly in immune-compromised patients. Shikonin is a well-known natural naphthazarin derivative with promising antifungal efficacy, but it's mechanism of action is still unclear. Keeping this in view, present work was designed to get a mechanistic insight of anti-candida efficacy of shikonin via in vitro experiments and in situ molecular modelling studies. The current exploratory study is based on research that uses both qualitative and quantitative techniques, including minimum inhibitory concentration, minimum biofilm inhibitory concentration, time kill assay, cell cycle analysis and apoptotic assays, static biofilm formation assays, microscopic biofilm assessment assays, ergosterol content estimation and molecular docking/simulation studies. The study revealed a notable effect of shikonin against Candida albicans, including retardation of biofilms. Shikonin, with its increasing concentration leads to candidal cell apoptosis and necrosis establishing its dose-dependent effect. Additionally, it exhibited fungicidal activity via a mechanism of action likely related to ergosterol complexation which was further corroborated by molecular docking and simulation studies.

Topics: Antifungal Agents; Biofilms; Candida; Candida albicans; Ergosterol; Humans; Microbial Sensitivity Tests; Molecular Docking Simulation; Naphthoquinones

Small cell lung cancer (SCLC) is a subtype of lung cancer with a very poor overall survival rate due to its extremely high proliferation and metastasis predilection. Shikonin is an active ingredient extracted from the roots of Lithospermum erythrorhizon, and exerts multiple anti-tumor functions in many cancers. In the present study, the role and underlying mechanism of shikonin in SCLC were investigated for the first time. We found that shikonin effectively suppressed cell proliferation, apoptosis, migration, invasion, and colony formation and slightly induced apoptosis in SCLC cells. Further experiment indicated the shikonin could also induced ferroptosis in SCLC cells. Shikonin treatment effectively suppressed the activation of ERK, the expression of ferroptosis inhibitor GPX4, and elevated the level of 4-HNE, a biomarker of ferroptosis. Both total ROS and lipid ROS were increased, while the GSH levels were decreased in SCLC cells after shikonin treatment. More importantly, our data identified that the function of shikonin was dependent on the up-regulation of ATF3 by performing rescue experiments using shRNA to silence the expression of ATF3, especially in the total and lipid ROS accumulaiton. Xenograft model was established using SBC-2 cells, and the results revealed that shikonin also significantly inhibited tumor growth by inducing ferroptosis. Finally, our data further confirmed that shikonin activated ATF3 transcription by impairing the recruitment of HDAC1 mediated by c-myc on the ATF3 promoter, and subsequently elevating of histone acetylation. Our data documented that shikonin suppressed SCLC by inducing ferroptosis in a ATF3-dependent manner. Shikonin upregulated the expression of ATF3 expression via promoting the histone acetylation by inhibiting c-myc-mediated HDAC1 binding on ATF3 promoter.

Topics: Activating Transcription Factor 3; Cell Line, Tumor; Ferroptosis; Histones; Humans; Lipids; Lung Neoplasms; Naphthoquinones; Reactive Oxygen Species; Small Cell Lung Carcinoma

In diabetes mellitus, diabetic nephropathy (DN) is a typical complication and pivotal cause of chronic kidney disease. The DN disease burden is among the highest in the world and is associated with high morbidity, mortality, and disease burden. Safe and effective medications are urgently needed for the treatment of DN. Interest has been increasing in Shikonin, extracted from the naphthoquinone plant, particularly in determining its renal protective effect.. In this study, we explored Shikonin's effects and potential mechanisms on a streptozotocin (STZ)-induced DN experimental model. An STZ-induced rat diabetic model was established, and the rats were treated with different doses of Shikonin (10/50 mg/kg) for 4 weeks. Blood, urine, and renal tissue samples were collected after the last administration. Renal tissues were examined to detect each group's physiologic, biochemical, histopathologic, and molecular changes.. The results showed that Shikonin administration could significantly alleviate the STZ-induced elevation of blood urea nitrogen, serum creatinine, urinary protein content, and renal pathologic injury. Furthermore, Shikonin significantly decreased oxidative stress, inflammation, and Toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor-κB expression levels in DN kidney tissues. Shikonin showed a dose-dependent effect, with the best outcome at 50 mg/kg.. Shikonin could effectively alleviate DN-related nephropathy damage and reveal the underlying pharmacologic mechanism. Based on the results, a Shikonin combination can be used in clinical treatment.

Topics: Albuminuria; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blood Urea Nitrogen; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Male; Naphthoquinones; Oxidative Stress; Rats; Rats, Sprague-Dawley; Streptozocin

Melanoma treatment is highly resistant to current chemotherapeutic agents. Due to its resistance towards apoptotic cell death, non-apoptotic cell death pathways are sought after.. We investigated a Chinese herbal medicine, shikonin, and its effect on B16F10 melanoma cells in vitro.. Cell growth of B16F10 melanoma cells treated with shikonin was analyzed using an MTT assay. Shikonin was combined with necrostatin, an inhibitor of necroptosis; caspase inhibitor; 3-methyladenine, an inhibitor of autophagy; or N-acetyl cysteine, an inhibitor of reactive oxygen species. Flow cytometry was used to assess types of cell death resulting from treatment with shikonin. Cell proliferation was also analyzed utilizing a BrdU labeling assay. Monodansylcadaverine staining was performed on live cells to gauge levels of autophagy. Western blot analysis was conducted to identify specific protein markers of necroptosis including CHOP, RIP1, and pRIP1. MitoTracker staining was utilized to identify differences in mitochondrial density in cells treated with shikonin.. Analysis of MTT assays revealed a large decrease in cellular growth with increasing shikonin concentrations. The MTT assays with necrostatin, 3-methyladenine, and N-acetyl cysteine involvement, suggested that necroptosis, autophagy, and reactive oxygen species are a part of shikonin's mechanism of action. Cellular proliferation with shikonin treatment was also decreased. Western blotting confirmed that shikonin-treated melanoma cells increase levels of stress-related proteins, e.g., CHOP, RIP, pRIP.. Our findings suggest that mainly necroptosis is induced by the shikonin treatment of B16F10 melanoma cells. Induction of ROS production and autophagy are also involved.

Topics: Apoptosis; Cell Line, Tumor; Cysteine; Humans; Melanoma; Naphthoquinones; Necrosis; Reactive Oxygen Species

Shikonin, a natural naphthoquinone compound, has a wide range of pharmacological effects, but its anti-tumor effect and underlying mechanisms in bladder cancer remain unclear.. We aimed to investigate the role of shikonin in bladder cancer in vitro and in vivo in order to broaden the scope of shikonin's clinical application.. We performed MTT and colony formation to detect the inhibiting effect of shikonin on bladder cancer cells. ROS staining and flow cytometry assays were performed to detect the accumulation of ROS. Western blotting, siRNA and immunoprecipitation were used to evaluate the effect of necroptosis in bladder cancer cells. Transmission electron microscopy and immunofluorescence were used to examine the effect of autophagy. Nucleoplasmic separation and other pharmacological experimental methods described were used to explore the Nrf2 signal pathway and the crosstalk with necroptosis and autophagy. We established a subcutaneously implanted tumor model and performed immunohistochemistry assays to study the effects and the underlying mechanisms of shikonin on bladder cancer cells in vivo.. The results showed that shikonin has a selective inhibitory effect on bladder cancer cells and has no toxicity on normal bladder epithelial cells. Mechanically, shikonin induced necroptosis and impaired autophagic flux via ROS generation. The accumulation of autophagic biomarker p62 elevated p62/Keap1 complex and activated the Nrf2 signaling pathway to fight against ROS. Furthermore, crosstalk between necroptosis and autophagy was present, we found that RIP3 may be involved in autophagosomes and be degraded by autolysosomes. We found for the first time that shikonin-induced activation of RIP3 may disturb the autophagic flux, and inhibiting RIP3 and necroptosis could accelerate the conversion of autophagosome to autolysosome and further activate autophagy. Therefore, on the basis of RIP3/p62/Keap1 complex regulatory system, we further combined shikonin with late autophagy inhibitor(chloroquine) to treat bladder cancer and achieved a better inhibitory effect.. In conclusion, shikonin could induce necroptosis and impaired autophagic flux through RIP3/p62/Keap1 complex regulatory system, necroptosis could inhibit the process of autophagy via RIP3. Combining shikonin with late autophagy inhibitor could further activate necroptosis via disturbing RIP3 degradation in bladder cancer in vitro and in vivo.

Topics: Autophagy; Cell Death; Humans; Kelch-Like ECH-Associated Protein 1; Naphthoquinones; Necroptosis; NF-E2-Related Factor 2; Reactive Oxygen Species; Urinary Bladder Neoplasms

Epstein-Barr virus (EBV) is a highly prevalent human herpesvirus that persists for life in more than 95% of the adult population. EBV usually establishes an asymptomatic life-long infection, but it is also associated with malignancies affecting B lymphocytes and epithelial cells mainly. The virus alternates between a latent phase and a lytic phase, both of which contribute to the initiation of the tumor process. So far, there is only a limited number of antiviral molecules against the lytic phase, most of them targeting viral replication. Recent studies provided evidence that EBV uses components of the NLRP3 inflammasome to enter the productive phase of its cycle following activation in response to various stimuli. In the present work, we demonstrate that shikonin, a natural molecule with low toxicity which is known to inhibit inflammasome, can efficiently repress EBV reactivation. Similar results were obtained with apigenin and OLT 1177, two other NLRP3 inflammasome inhibitors. It is shown herein that shikonin repressed the transcription of reactivation-induced NLRP3 thereby inhibiting inflammasome activation and EBV lytic phase induction.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apigenin; Cell Line; Cell Line, Tumor; Herpesvirus 4, Human; Humans; Inflammasomes; Naphthoquinones; NLR Family, Pyrin Domain-Containing 3 Protein; Virus Activation

Glioblastoma is a type of intracranial malignancy. Shikonin, a Chinese traditional medicine, has been shown to have anti-tumor efficacy toward human glioblastoma cells in vitro. However, shikonin cannot easily cross the blood-brain barrier. To address this issue, we evaluated the anti-tumor effects of direct intracranial infusion of shikonin in in vivo orthotopic syngeneic murine glioblastoma models using C57BL/6 mice.. The cytotoxic effects of shikonin against murine glioblastoma cells, SB28 and CT-2A, were reported resistance to temozolomide, were evaluated using an allophycocyanin-conjugated annexin V and propidium iodide assay with flow cytometry. Impedance-based real-time cell analysis (RTCA) was used to analyze the inhibitory effects of shikonin on growth and proliferation. To evaluate the anti-tumor activity of shikonin in vivo, we used orthotopic syngeneic murine glioblastoma models with SB28 and CT-2A cells.. In flow cytometry-based cytotoxic assays, shikonin induced apoptosis. RTCA indicated that shikonin decreased the cell index of murine glioblastoma cells, SB28 and CT-2A, in a dose-dependent manner (p < 0.0001 for both cell lines), while temozolomide did not (p = 0.91 and 0.82, respectively). In murine glioblastoma models, SB28 and CT-2A, direct intracranial infusion of shikonin, as a local chemotherapy, improved the overall survival of mice in a dose-dependent manner compared with control groups (p < 0.0001 and p = 0.02, respectively). While temozolomide did not (p = 0.48 and 0.52, respectively).. The direct intracranial infusion of shikonin has potential as a local therapy for patients with glioblastoma.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Glioblastoma; Humans; Mice; Mice, Inbred C57BL; Naphthoquinones; Temozolomide

Microwave (MW) thermal therapy has been widely used for the treatment of cancer in clinics, but it still shows limited efficacy and a high recurrence rate owing to non-selective heat delivery and thermo-resistance. Regulating glycolysis shows great promise to improve MW thermal therapy since glycolysis plays an important role in thermo-resistance, progression, metabolism, and recurrence. Herein, we developed a delivery nanosystem of shikonin (SK)-loaded and hyaluronic acid (HA)-modified hollow Fe-MOF (HFM), HFM@SK@HA, as an efficient glycolysis-meditated agent to improve the efficacy of MW thermal therapy. The HFM@SK@HA nanosystem shows a high SK loading capacity of 31.7 wt %. The loaded SK can be effectively released from the HFM@SK@HA under the stimulation of an acidic tumor microenvironment and MW irradiation, overcoming the intrinsically low solubility and severe toxicity of SK. We also find that the HFM@SK@HA can not only greatly improve the heating effect of MW in the tumor site but also mediate MW-enhancing dynamic therapy efficiency by catalyzing the endogenous H

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Female; Hep G2 Cells; Humans; Iron; L Cells; Metal Nanoparticles; Metal-Organic Frameworks; Mice; Microwaves; Naphthoquinones; Neoplasms; Warburg Effect, Oncologic

Topics: Animals; Heat Stroke; Interleukin-17; Mice; Naphthoquinones; Oxidative Stress

Naturally occurring naphthoquinones, shikonin and alkannin, are important ingredients of traditional Chinese medicine Zicao. These constituents are reported to have many therapeutic uses, such as wound healing; scar treatment; and anti-inflammation, anti-acne, anti-ulcer, anti-HIV, anticancer, and antibacterial properties. The primary objective of this investigation was to explore the effect of shikonin and alkannin on Escherichia coli ATP synthase and its cell growth. Shikonin caused complete (100 %) inhibition, and alkannin caused partial (79 %) inhibition of wild-type E. coli ATP synthase. Both caused partial (4 %-27 %) inhibition of ATP synthase with genetically modified phytochemical binding site. The growth inhibition of strains expressing normal, deficient, and mutant ATP synthase by shikonin and alkannin, corroborated the inhibition observed in isolated normal wild-type and mutant ATP synthase. Trivial inhibition of mutant enzymes indicated αR283D, αE284R, βV265Q, and γT273A are essential for formation of the phytochemical binding site where shikonin and alkannin bind. Further, shikonin was a potent inhibitor of ATP synthase than alkannin. The antimicrobial properties of shikonin and alkannin were tied to the binding at phytochemical site of microbial ATP synthase. Selective targeting of bacterial ATP synthase by shikonin and alkannin may be an advantageous alternative to address the antibiotic resistance issue.

Topics: Adenosine Triphosphate; Escherichia coli; Naphthoquinones; Phytochemicals

Myxofibrosarcoma (MFS) is a subtype of soft tissue sarcoma of connective tissue, which is characterized by large intra-tumor heterogeneity. Therapy includes surgical resection. Additional chemotherapy is of limited effect. In this study, we demonstrated the potent anticancer activity of shikonin derivatives in our MFS cellular model of tumor heterogeneity for developing a new therapeutic approach. The impact of shikonin and β,β-dimethylacrylshikonin (DMAS) on viability, apoptotic induction, MAPK phosphorylation, and DNA damage response were analyzed by means of two human MFS cell lines, MUG-Myx2a and MUG-Myx2b, derived from a singular tumor tissue specimen. MFS cells showed a dose-dependent inhibition of cell viability and a significant induction of apoptosis. Treatment with shikonin derivatives caused an inhibition of pSTAT3 and an increase in pAKT, pERK, pJNK, and pp38. DMAS and shikonin inhibited the activation of the two master upstream regulators of the DNA damage response, ATR and ATM. MUG-Myx2b, which contains an additional

Topics: Adult; Apoptosis; Cell Line, Tumor; Fibrosarcoma; Humans; Naphthoquinones; Signal Transduction

Alkannin is the main coloring matter of Alkanet, a natural red dye extracted from the root of Alkanna tinctoria L. Shikonin, the optical isomer of alkannin, is extracted from Lithospermum erythrorhizon. As both red dyes are only slightly soluble in water, the application of ordinary Raman spectroscopy is limited. Thus, Surface-enhanced Raman spectroscopy (SERS) can be successfully applied to the study of the red dyes solutions. Solid alkannin and shikonin were characterized by ordinary Raman spectroscopy. Density Functional Theory (DFT) methods were used to calculate the Raman spectrum of the dyes and to assign the experimental Raman bands to their vibrational normal modes. Different pH conditions were tested in order to determine the optimal conditions for the SERS detection of alkannin and shikonin. Based on the previous results, a perpendicular orientation of the red dyes on the Ag substrate was deducted. Finally, shikonin was identify by SERS spectroscopy in a dyed paper sample from an 8th century handscroll from Japan.

Topics: Coloring Agents; Japan; Naphthoquinones; Spectrum Analysis, Raman

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (M

Topics: Antiviral Agents; Catalytic Domain; Coronavirus; Coronavirus 3C Proteases; Crystallography, X-Ray; Molecular Docking Simulation; Naphthoquinones; Protease Inhibitors; Protein Binding

Most of the antitumor chemotherapeutic drugs execute the therapeutic performance upon eliciting tumor cell apoptosis, which may cause chemoresistance of tumors. Design of novel drugs to eradicate apoptosis-resistant tumors via non-apoptotic cell death pathways is promising for improving the long-term chemotherapeutic efficacy. Herein, a Fe(III)-Shikonin metal-polyphenol-coordinated supramolecular nanomedicine for combined therapy of tumor via ferroptosis and necroptosis is designed. The construction of the nanomedicine based on the coordinated self-assembly between Fe

Topics: Cell Line, Tumor; Ferric Compounds; Ferroptosis; Humans; Nanomedicine; Naphthoquinones; Necroptosis

Shikonin (SKN), a naturally occurring naphthoquinone, is a major active chemical component isolated from Lithospermum erythrorhizon Sieb Zucc, Arnebia euchroma (Royle) Johnst, or Arnebia guttata Bunge, and commonly used to treat viral infection, inflammation, and cancer. However, its underlying mechanism has not been elucidated.. This study aims to explore the antitumor mechanism of SKN in colorectal cancer (CRC) through network pharmacology and cell experiments.. SymMap database and Genecards were used to predict the potential targets of SKN and CRC, while the cotargets were obtained by Venn diagram. The cotargets were imported into the website of String and DAVID, constructing the protein-protein interaction (PPI) network, performing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, the Compound-Target-Pathway (C-T-P) network was generated by connecting potential pathways with the corresponding targets.. According to the results of network pharmacological analysis, the cell experiments were used to verify the key signal pathway. The most relevant target of SKN for the treatment of CRC was PI3K/Akt signaling pathway. SKN inhibited CRC cells (HT29 and HCT116) proliferation, migration, and invasion, and promoted cell apoptosis by targeting IL6 and inhibiting the IL6R/PI3K/Akt signaling pathway. SKN promotes apoptosis and suppresses CRC cells' (HT29 and HCT116) activity through the PI3K-Akt signaling pathway.. This research not only provided a theoretical and experimental basis for more in- -depth studies but also offered an efficient method for the rational utilization of a series of Traditional Chinese medicines as anti-CRC drugs.

Topics: Colorectal Neoplasms; Drugs, Chinese Herbal; Humans; Molecular Docking Simulation; Naphthoquinones; Network Pharmacology; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt

Shikonin, a naturally occurring naphthoquinone with potent anti-tumor activity, has been reported to induce cancer cell death via targeting selenoenzyme thioredoxin reductase 1 (TrxR1; TXNRD1). However, the interaction between shikonin and TrxR1 remains unclear, and the roles of the cellular antioxidant system in shikonin induced cell death are obscure. Here, we found that shikonin modified the Sec

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Kelch-Like ECH-Associated Protein 1; Lung Neoplasms; Naphthoquinones; Necroptosis; NF-E2-Related Factor 2; Oxidation-Reduction; Oxygen; Reactive Oxygen Species; Thioredoxin Reductase 1; Thioredoxin-Disulfide Reductase

Although chemoimmunotherapy has achieved considerable success in cancer treatment in recent years, the cure for triple-negative breast cancer (TNBC) remains elusive. The unsatisfied outcomes are likely attributed to deficient tumor immunogenicity, a strong immunosuppressive tumor microenvironment (ITM) and tumor metastasis. To address this issue, we constructed an effective codelivery system, combined with tumor growth factor β (TGF-β) small interference RNA (siTGF-β) and shikonin (SK), to achieve successful chemoimmunotherapy of TNBC. The SK/siTGF-β NPs (approximately 110 nm) exhibited a uniform structure and good stability. Conjugated FA presented enhanced cellular uptake in 4T1 cells, and siTGF-β escaped from lysosomes because of the "proton sponge" effect of PEI. Furthermore, SK actually induced satisfactory immunogenic cell death (ICD) and the resulting dendritic cell (DC) maturation facilitated a distinctly enhanced cytotoxic T lymphocyte (CTL) response, generating a positive effect on tumor suppression. Simultaneously, the successful silencing of TGF-β alleviated the TGF-β-mediated ITM and inhibited the epithelial-to-mesenchymal transition (EMT), contributing to the infiltration of CTLs, suppression of regulatory T lymphocyte (Treg) proliferation and lung metastasis inhibition. Thus, the SK/siTGF-β NPs demonstrated the strongest therapeutic effect with delayed tumor growth (TIR = 88.5%) and lung metastasis restraint (77.3%). More importantly, tumor rechallenge assay suggested that the codelivery system produced a long-term immunological memory response to prevent tumor recurrence. Based on boosting the immune response and combating the ITM, SK/siTGF-β NPs would be a potential approach for TNBC therapy.

Topics: Cell Line, Tumor; Humans; Immunotherapy; Naphthoquinones; Triple Negative Breast Neoplasms; Tumor Microenvironment

Tauopathies such as Alzheimer's and Parkinson's diseases involve the abnormal deposition of tau aggregates in the brain and neuronal tissues. We report that a natural naphthoquinone, shikonin, impeded the oligomerization and fibrillization of tau. The compound strongly inhibited heparin, arachidonic acid, and RNA-induced tau aggregation. Atomic force microscopy, dynamic light scattering, SDS-PAGE, and dot blot assays revealed that shikonin diminished tau oligomerization and decreased the mean size of tau oligomers. Transmission electron microscopy and atomic force microscopy analysis further showed that shikonin could suppress tau fibrillization and shorten the tau filaments. Shikonin inhibited tau droplet formation. The compound significantly reduced the aggregation rate of a tryptophan mutant (Y310W-tau) of tau. In addition, shikonin disaggregated preformed tau filaments with a half-maximal disaggregation concentration (DC

Topics: Alzheimer Disease; Humans; Naphthoquinones; Neurons; tau Proteins; Tauopathies

Osteoarthritis (OA) is a chronic joint degenerative disease characterised by narrowed articular space, formation of surrounding osteophytes, and subchondral bone sclerosis. OA is caused by cartilage degeneration, which is closely correlated with the disequilibrium of anabolism and catabolism in chondrocytes. Previous studies have revealed that autophagy plays a significant role in maintaining the balance of anabolic and catabolic activities. Thus, targeting autophagy may be a promising therapeutic strategy for OA. Shikonin, a traditional Chinese herbal medicine isolated from flavonoid glucuronide, has drawn focus for its role in activating autophagy. In this study, the mRNA and protein level of a disintegrin and metalloproteinase with thrombospondin motifs-5 and matrix metalloproteinases-1 decreased with shikonin treatment, in the IL-1β-induced OA cell model. On the contrary, IL-1β-induced downregulation of Aggrecan and Collagen II was ameliorated following shikonin treatment. In addition, the upregulation of autophagy-related marker genes Beclin-1 and LC3II/LC3I in chondrocytes indicated that autophagy could be activated upon shikonin treatment. Moreover, shikonin's promotion of anabolism in chondrocytes through autophagy activation corresponded with the results from the examination using chloroquine, an autophagy inhibitor. OA mouse cartilage tissues were stained with safranin O and fast green dyes. Results were analysed using the Osteoarthritis Research Society International (OARSI) score, and suggested that mice cartilage degeneration was alleviated after shikonin treatment. Altogether, we identified that shikonin might be a novel promising drug for OA treatment.

Topics: Animals; Autophagy; Cartilage, Articular; Cells, Cultured; Chondrocytes; Interleukin-1beta; Mice; Naphthoquinones; Osteoarthritis

Topics: Autophagy; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; GTP-Binding Protein beta Subunits; Humans; MicroRNAs; Naphthoquinones

Allergic rhinitis (AR) is a disease in the nasal mucosa related with Th2 lymphocyte inflammatory action. Dendritic cells (DCs) have been proved that they played a significant role in the development and maintenance of AR. However, there is still a lack of specific therapies for DCs in clinical practice. Shikonin (SHI) is a natural naphthoquinone compound isolated from the Chinese herb Radix Arnebiae. It is reported that SHI can interference the phenotype and function of dendritic cells, so we speculate that SHI may be an effective drug for the treatment of AR. However, the clinical usage of SHI has been limited by the bioactive properties of poor solubility, short retention time and low bioavailability. Therefore, in order to better exert the anti-inflammatory effect of SHI, an efficient SHI delivery system is urgently needed.. We prepared and characterized SHI-PM and NGR-SHI-PM with the thin-film hydration method. We used retrodialysis method to explore the release behavior. We took immunofluorescence to investigate the expression of CD13 in vitro. Then we tested BM-DCs mature cell detection by flow cytometry. An allergic rhinosinusitis murine model, hematoxylin and eosin stain and flow cytometry were established to test the efficiency of anti-inflammation in vivo. At last, western blot analysis and plasmid construction and transfection assay were taken to reveal the molecular mechanisms.. In the present study, we revealed that NGR-modifified could strengthen the intracellular uptake of PM (p < 0.001) and CD13 was high expressed on mature BM-DCs (p < 0.001). NGR-modified could enhance the inhibition of SHI in vitro (p < 0.05). NGR-modifified could increase the distribution of PM in vivo by DiI fluorescently (p < 0.01). NGR-modified could enhance SHI anti-allergic activity in OVA-sensitized mice and enhance the inhibition of SHI on DC maturation in lymph node (p < 0.001). Our findings also suggest that SHI may have the inhibitory effect on AR through NF-κB pathway by targeting PARP.. In summary, we have shown that NGR-PM-SHI could be a novel strategy for targeted treating allergic rhinitis through the NF-κB pathway by targeting PARP.

Topics: Animals; Dendritic Cells; Disease Models, Animal; Mice; Mice, Inbred BALB C; Micelles; Naphthoquinones; Nasal Mucosa; NF-kappa B; Ovalbumin; Poly(ADP-ribose) Polymerase Inhibitors; Polyesters; Polyethylene Glycols; Rhinitis, Allergic

Shikonin(SK) is a natural small molecule naphthoquinone compound, which has anti-cancer activity in various human malignant tumors. Pyrroline-5-carboxylate reductase 1(PYCR1) is involved in tumorigenesis and regulates various cellular processes, including growth, invasion, migration, and apoptosis. However, the effect of SK and PYCR1 on apoptosis and autophagy in hepatocellular carcinoma are unclear. Our goal is to determine the internal molecular mechanism of the interaction between SK and PYCR1 and its role in the occurrence and development of liver cancer. The CCK8 assay, wound healing assay, and transwell assays show that SK and siPYCR1(gene silence PYCR1) inhibited the malignant phenotype of HCC cells, including cell viability, colony formation, migration, and invasion, respectively. The flow cytometry assays and immunofluorescence show that SK and siPYCR1 activated apoptosis and autophagy, respectively. SK induces apoptosis and autophagy in a dose-dependent manner. In addition, HCC cells were transfected with small interference fragment PYCR1 siRNA to construct siPYCR1 and SK single treatment group and co-treatment group to verify the interaction between SK and PYCR1. The Western blot identified that PI3K/Akt/mTOR signal pathway protein expression was significantly downregulated in HCC cells treated with SK and siPYCR1 together. Collectively, SK may induce apoptosis and autophagy by reducing the expression of PYCR1 and suppressing PI3K/Akt/mTOR. Thus, SK may be a promising antineoplastic drug in Hepatocellular carcinoma (HCC). SK downregulating PYCR1 might supply a theoretical foundation for the potential therapeutic application in hepatocellular carcinoma.

Topics: Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Humans; Liver Neoplasms; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyrroles; Pyrroline Carboxylate Reductases; TOR Serine-Threonine Kinases

This study reports the formulation and delivery of hyaluronic acid-Zein (HA-Zein) nanogels loaded with Shikonin (SK) to selectively attenuate macrophage inflammasome. The self-assembled nanogels, produced by nanoprecipitation, exhibited high encapsulation efficiency, and were selectively internalized by human THP-1-derived macrophages without eliciting cytotoxic responses. Cell treatment with HA-Zein-SK nanogels before stimulation with LPS and Nigericin significantly suppressed caspase-1 activation and IL-1β production, indicating inflammasome inhibition. Importantly, HA-Zein-SK nanogels bioinstructed inflammasome activated macrophages towards an anti-inflammatory CD163

Topics: Inflammasomes; Interleukin-1beta; Macrophages; Nanogels; Naphthoquinones; NLR Family, Pyrin Domain-Containing 3 Protein; Zein

Topics: Cartilage, Articular; Cells, Cultured; Chondrocytes; Humans; Inflammation; Naphthoquinones; Osteoarthritis

Natural compounds were used in the treatment of acute kidney injury (AKI) caused by sepsis. This study investigated the function of shikonin from the roots of Arnebia purpurea in sepsis-induced AKI model. The target genes of shikonin were predicted by traditional Chinese medicine integrative database (TCMID). The markers of kidney injury, oxidative stress, and inflammatory factors were measured by enzyme-linked immunosorbent assay (ELISA). The pathological changes of kidney tubules were assessed by Hematoxylin and Eosin staining. Apoptosis of kidney tubular epithelial cells (KTECs) was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Protein expression was measured by western blot. Shikonin significantly improved kidney injury induced by cecal ligation and perforation (CLP). Besides, shikonin reduced KTECs apoptosis, malondialdehyde (MDA), reactive oxygen species (ROS), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) levels, while augmented SOD and IL-10 levels. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase4 (NOX4) was predicted a target gene of shikonin. The expression of NOX4 was significantly inhibited in shikonin-treated group and the levels of phosphatidylinositol 3,4,5-trisphosphate 3-phosphate and dual specificity protein phosphate (PTEN) and p-p65 were decreased, while level of p-Akt was elevated. In vitro experiments, shikonin inhibited cell apoptosis, inflammatory, and ROS in human HK-2 cells and rat TECs. Shikonin downregulated expression of NOX4, PTEN and p-p65, and upregulated p-AKT and Bcl-2 expression in HK2 cells treated with lipopolysaccharide (LPS). Moreover, overexpression of NOX4 enhanced the effect of LPS on the expression level of PTEN, p-p65, p-AKT, and Bcl-2, which was reversed by the addition of shikonin. Taken together, shikonin could improve sepsis-induced AKI in rats, and attenuate the LPS induced KTECs apoptosis, oxidative stress, and inflammatory reaction via modulating NOX4/PTEN/AKT pathway.

Topics: Acute Kidney Injury; Animals; Apoptosis; Epithelial Cells; Humans; Kidney; Lipopolysaccharides; NADPH Oxidase 4; Naphthoquinones; Oxidative Stress; Phosphates; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Rats; Reactive Oxygen Species; Sepsis

Shikonin is an ointment produced from Lithospermun erythrorhizon which has been used in traditional medicine both in Europe and Asia for wound healing and is associated with anti-inflammatory properties. The goal of this work is to assess the analgesic properties of Shikonin in the CFA-induced inflammation model of pain. Rats were subjected to inflammation of the hind paw by CFA injection with a preventive injection of Shikonin and compared to either a control group or to a CFA-inflamed group with the vehicle drug solution. Inflammation of the hind paw by CFA was assessed by measurement of the dorsal to plantar diameter. Mechanical thresholds were established by means of the Von Frey filaments which are calibrated filaments that exert a defined force. Finally, the spinal cord of the studied animals was extracted to analyse the microglia population through immunohistochemistry using the specific marker Iba-1. Our results show that Shikonin reduces the paw oedema caused by CFA inflammation. Subsequently, there is a concomitant restoration of the mechanical thresholds reduced by CFA hind paw injection. Additionally, spinal microglia is activated after CFA-induced inflammation. Our results show that microglia is inhibited by Shikonin and has concomitant restoration of the mechanical thresholds. Our findings demonstrate for the first time that Shikonin inhibits microglia morphological changes and thereby ameliorates pain-like behaviour elicited by mechanical stimulation.

Topics: Animals; Disease Models, Animal; Hyperalgesia; Inflammation; Microglia; Naphthoquinones; Pain; Rats; Spinal Cord

Topics: Animals; Imiquimod; Lipopolysaccharides; Macrophages; Methotrexate; Mice; Naphthoquinones; Psoriasis

Shikonin is a natural multipotent anti-tumorigenic compound. We investigated potential synergy between shikonin and anti-diabetic metformin against tumorigenic properties of breast cancer cell line MCF-7.. Shikonin and metformin synergize in inhibiting the tumorigenic activities of MCF-7 cells including their proliferation, invasiveness, and EMT with a potential to inhibit multidrug resistance.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Metformin; Naphthoquinones

Shikonin is widely acknowledged as a bioactive substance extracted from the root of lithospermum erythrorhizon with multifunction. It alleviates ischemic/reperfusion (I/R) injury in liver and brain. Due to the similar pathogenesis of I/R and hypoxia/reoxygenation (H/R)-stimulated injury, we aimed to explore the potential pharmacological effects of Shikonin on the myocardial injury. The rats with myocardial I/R injury and the primary cardiomyocytes with H/R-stimulated injury were taken as in vivo and in vitro models. 2,3,5-Triphenyltetrazolium chloride staining and ELISA kits were used for detection of myocardial infarction and cardiac injury. Hematoxylin and eosin and immunohistochemistry staining were used to analyze the effect of Shikonin on autophagy histology. Western blot was performed to detect the proteins related to autophagy and Hippo pathway. The results showed that SHK reduces the size of myocardial infarction, improved cardiac function, suppressed the expression of autophagy-related proteins, and reduced the amount of autophagosomes. The underlying mechanism is to activate Hippo pathway. In vitro assay also suggested that SHK enhanced the cell viability, reduced the apoptotic rates in rat primary cardiomyocytes. Collectively, our results demonstrated that SHK protects against myocardial I/R injury by inhibiting autophagy, of which the underlying molecular mechanism is to activate the Hippo signaling pathway.

Topics: Animals; Apoptosis; Autophagy; Hippo Signaling Pathway; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Naphthoquinones; Rats; Signal Transduction

Shikonin is the main component of root extracts from the Chinese herbal medicine

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Molecular Docking Simulation; Naphthoquinones; p21-Activated Kinases; Pancreatic Neoplasms

Melanoma is a complex and heterogenous disease, displays the deadliest form of skin cancer, and accounts for approx. 80% of all skin cancer deaths. In this study, we reported on the synthesis and pharmacological effects of a novel shikonin derivative (SK119), which is active in a nano-molar range and exhibits several promising in vitro effects in different human melanoma cells. SK119 was synthesized from shikonin as part of our search for novel, promising shikonin derivatives. It was screened against a panel of melanoma and non-tumorigenic cell lines using XTT viability assays. Moreover, we studied its pharmacological effects using apoptosis and Western blot experiments. Finally, it was combined with current clinically used melanoma therapeutics. SK119 exhibited IC

Topics: Apoptosis; Azetidines; Cell Line; Humans; Melanoma; Naphthoquinones; Piperidines; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Vemurafenib

Diabetes is a complicated multifactorial disorder in which the patient generally observes polyphagia, polydipsia, and polyuria due to uncontrolled growth in blood sugar levels. For its management, the pharmaceutical industry is working day and night to find a better drug with no or least toxicity. That's why nowadays a more focused branch is to use herbal phytoconstituents for its prevention. Shikonin is a naphthoquinone natural dye that is isolated from the plants of the Boraginaceae family and has proven its role as an anti-cancer, anti-inflammatory, and anti-gonadotrophic agent. In our previous study, we have published its anti-diabetic action by inhibiting the enzyme protein tyrosine phosphatase 1B. In this study, we were more focused on finding out the role of Shikonin and its pharmacophores by inhibiting the action of aldose reductase (AR) enzyme. The study was conducted using pharmacophore modeling, molecular docking, and molecular dynamics simulation studies. The absorption, distribution, metabolism, excretion (ADME), and toxicity profile were also evaluated in this study. Along with all the computational biology parameters we also focused on the in vitro activity and kinetic study of inhibitory activity of Shikonin against aldose reductase.

Topics: Aldehyde Reductase; Diabetes Mellitus; Enzyme Inhibitors; Humans; Hypoglycemic Agents; Molecular Docking Simulation; Naphthoquinones

Glioblastoma multiforme is a malignant neoplasia with a median survival of less than two years and without satisfactory therapeutic options. The so-called glioblastoma stem cells escape the established radio- and chemotherapies and lead to tumor recurrence in most cases. The alkaloid Shikonin with its various anti stem cell properties and the interstitial photodynamic therapy with 5-aminolevulinic acid seem to be promising new options in the therapy of glioblastoma. In this study, in vitro investigations were performed to observe the influence of Shikonin on viability, proliferation, induction of apoptosis and the capability of forming tumor spheres in U-87 MG and the primary glioblastoma cell line GB14. The combined effect with the chemotherapeutic temozolomide and photodynamic treatment on the mRNA expression of glioma specific stem cell markers and further examined intracellular protoporphyrin IX accumulation under Shikonin treatment was analyzed. Shikonin effectively inhibited the capability of forming tumor spheres and enhanced temozolomide effectiveness in the reduction of proliferation and in the induction of apoptosis. Additionally, Shikonin increased the mRNA expression of the tumor suppressing Neurofibromatosis type 1 (NF1) gene and showed modulating effects on intracellular protoporphyrin IX.

Topics: Aminolevulinic Acid; Brain Neoplasms; Cell Line, Tumor; Glioblastoma; Humans; Naphthoquinones; Neoplasm Recurrence, Local; Photochemotherapy; RNA, Messenger; Temozolomide

Albeit oxidative stress has been implied in the pathogenesis of tubal pregnancy (TP), there are scant data to suggest that ferroptosis occurs in TP. Shikonin plays a pivotal role in redox status, but whether it can regulate ferroptosis to treat TP remains unknown.. We collected and analyzed ferroptosis-related indices from the villous tissue (VT) of women suffering from TP and from women with a normal pregnancy. In vitro, we used shikonin and/or RAS-selective lethal 3 (RSL3) to intervene HTR-8/SVneo cells and further detected ferroptosis indices and cell functions. Finally, the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) is pharmacologically activated to explore the effect of Nrf2 on shikonin regulating ferroptosis.. This study firstly showed that ferroptosis may be involved in TP pathogenesis and shikonin potentially targeted ferroptosis to treat TP.

Topics: Carbolines; Cell Death; Female; Ferroptosis; Humans; Naphthoquinones; NF-E2-Related Factor 2; Phospholipid Hydroperoxide Glutathione Peroxidase; Pregnancy, Tubal; Reactive Oxygen Species

The mitochondria represent a potential target for the treatment of triple-negative breast cancer (TNBC) and shikonin (SK) has shown remarkable therapeutic effects on TNBC. Herein, it is found that SK possesses potent inhibitory effects on mitochondrial biogenesis via targeting polymerase gamma (POLG). However, its application is restricted by its poor aqueous solubility and stability, and therefore, a biomimetic micelle to aid with tumor lesion accumulation and mitochondria-targeted delivery of SK is designed. A folic acid (FA) conjugated polyethylene glycol derivative (FA-PEG-FA) is inserted onto the external membranes of red blood cells (FP-RBCm) to prepare a "right-side-out" RBCm-camouflaged cationic micelle (ThTM/SK@FP-RBCm). Both FP-RBCm coating and a triphenylphosphine (TPP) moiety on the periphery of micelles contribute to tumor lesion distribution, receptor-mediated cellular uptake, and electrostatic attraction-dependent mitochondrial targeting, thereby maximizing inhibitory effects on mitochondrial biosynthesis in TNBC cells. Intravenous administration of ThTM/SK@FP-RBCm leads to profound inhibition of tumor growth and lung metastasis in a TNBC mouse model with no obvious toxicity. This work highlights the mitochondria-targeted delivery of SK using a "right-side-out" membrane-camouflaged micelle for the inhibition of mitochondrial biogenesis and enhanced therapeutic effects on TNBC.

Topics: Animals; Cell Line, Tumor; Folic Acid; Humans; Mice; Micelles; Naphthoquinones; Organelle Biogenesis; Polyethylene Glycols; Triple Negative Breast Neoplasms

Although chondrosarcoma is the second most common primary malignant bone tumor, treatment options are limited due to its extensive resistance to a chemo- and radiation therapy. Since shikonin has shown potent anticancer activity in various types of cancer cells, it represents a promising compound for the development of a new therapeutic approach.. The dose-relationships of shikonin and its derivatives acetylshikonin and cyclopropylshikonin on two human chondrosarcoma cell lines were measured using the CellTiter-Glo®. The changes in the cell cycle were presented by flow cytometry. Protein phosphorylation and expression apoptotic markers, MAPKs and their downstream targets were analyzed using western blotting and gene expression were evaluated using RT-qPCR.. These data demonstrated the significant anti-tumorigenic effect of shikonin derivatives in chondrosarcoma and encourage further research.

Topics: Apoptosis; Bone Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Chondrosarcoma; Humans; Mitogen-Activated Protein Kinases; Naphthoquinones; Receptors, Death Domain

Methicillin-resistant

Topics: Anti-Bacterial Agents; Drug Synergism; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Naphthoquinones; Staphylococcus aureus

Ferroptosis has been considered as a promising pathway to overcome apoptosis-induced tumor chemoresistance. However, the antitumor efficacy of ferroptosis-inducing agents is still limited because of the complexity and diversity of tumor microenvironments. Herein, we demonstrate a triple ferroptosis amplification strategy for tumor therapy by associating iron-based nanocarriers, ferroptosis molecular drugs, and H

Topics: Cell Line, Tumor; Ferric Compounds; Ferroptosis; Glutathione; Hydrogen Peroxide; Nanomedicine; Naphthoquinones; Sorafenib

Colorectal cancer (CRC) and inflammatory bowel disease (IBD) are the most common diseases of human digestive system. Nowadays, the influence of the inflammatory microenvironment on tumorigenesis has become a new direction, and the exploration of relative molecular mechanism will facilitate the discovery and identification of novel potential anti-cancer molecules.. Natural shikonin (SK) and acetyl-shikonin (acetyl-SK) was administered to azoxymethane (AOM)/dextran sodium sulphate (DSS)-induced colitis-associated colorectal cancer (CAC) mice model by gavage to investigate their therapeutic effects. Moreover, fresh feces and colon tissues were collected for determining the function of SK and acetyl-SK on the gut microbes and protein expression, respectively.. Both SK and acetyl-SK decreased AOM/DSS-induced CAC, and regulated the intestinal flora structure in CAC mouse model. They, especially SK, improved species richness, evenness and diversity of intestinal flora, recovered the upregulated ratio of Firmicutes to Bacteroidota (F/B ratio) which symbolizes gut microbiota dysbiosis. SK and its derivative increased the beneficial bacteria g__norank_f__Muribaculaceae, Lactobacillus, Lachnospiraceae_NK4A136_Group, and reduced those harmful ones including Ileibacterium and Coriobacteriaceae UCG-002. Notably, AOM/DSS caused significant increase in the abundance of Ileibaterium valens and g__norank_f__norank_o__Clostridia_UCG-014, which were not previously reported in studies of colonic inflammation or cancer, and the disorder was reversed by 20 mg/kg of SK. In our current study, the action of SK and acetyl-SK is dose-dependent, and 20 mg/kg SK exhibited the most effective functions, even better than the positive drug mesalazine. Moreover, differential proteomics and ELISA results showed that SK could recover the increase of pro-inflammatory cytokines (including IL-1β, IL-6 and TNF-α), the upregulation of pyruvate kinase isozyme type M2 (PKM2) and some other proteins (mainly concentrated in transcriptional mis-regulation in cancer and IL-17 signaling pathways), and the downregulation of Aldh1b1-Acc3-Maoa and Μgt2b34-Aldh1a1-Aldh1a7 involved in Wnt/β-catenin signaling pathway.. Our study identified SK and acetyl-SK, especially SK, as potential preventive agents for CAC through regulating both gut microbes and pathways involved in inflammation and cancer such as Wnt/β-catenin signaling pathway.

Topics: Animals; Azoxymethane; Bacteroidetes; Colitis; Colitis-Associated Neoplasms; Colorectal Neoplasms; Dextran Sulfate; Disease Models, Animal; Firmicutes; Humans; Inflammation; Mice; Mice, Inbred C57BL; Naphthoquinones; Tumor Microenvironment

Shikonin, a naphthoquinone dye, is a molecule of colour of natural origin, whose peculiar properties have not yet been fully rationalized. Its core structure consists of a di-hydroxy-naphthoquinone with an additional non-aromatic hydroxy group. From a comprehensive study involving fast spectroscopic techniques (fs-TA and fs-UC) and TDDFT electronic structure calculations on shikonin (Shk) and its derivatives 5-hydroxy-1,4-naphthoquinone (5HNQ), 5,8-diacetoxy-1,4-naphthoquinone (DiAc), 5,8-dihidroxy-1,4-naphthoquinone (DHNQ) and acetylshikonin, AcShk, it is shown that intramolecular excited state proton transfer (ESIPT) is present and is determinant in the deactivation of the hydroxy containing molecules. This is mirrored by the dominance of the internal conversion deactivation channel. In Shk, the non-aromatic hydroxy group determines the preferred conformer in both the ground- and excited-state, as reflected in the doubling of the fluorescence quantum yield value of this molecule relative to DHNQ. From fs-UC, a kinetic isotopic effect of 1.7 was obtained for DHNQ.

Topics: Models, Molecular; Naphthoquinones; Protons; Quantum Theory

Shikonin induces glioma cell death via necroptosis, a caspase-independent programmed cell death pathway that is chiefly regulated by receptor-interacting serine/threonine protein kinase1 (RIP1) and 3 (RIP3). Chromatinolysis is considered as one of the key events leading to cell death during necroptosis. It is usually accompanied with nuclear translocation of AIF and formation of γ-H2AX. Cyclophilin A (CypA) is reported to participate in the nuclear translocation of AIF during apoptosis. However, it remains unclear whether CypA contributes to necroptosis and regulation of chromatinolysis. In this study, our results revealed for the first time that shikonin promoted time-dependent CypA activation, which contributed to nuclear translocation of AIF and γ-H2AX formation. In vitro studies showed that knockdown of CypA by siRNA or inhibition of CypA by its specific inhibitor, cyclosporine A (CsA), not only significantly mitigated shikonin-induced glioma cell death, but also prevented chromatinolysis. Mechanistically, activated CypA targeted mitochondria and triggered mitochondrial superoxide overproduction, which then promoted AIF translocation from mitochondria into the nucleus by depolarizing the mitochondria and intensified the formation of γ-H2AX by promoting intracellular accumulation of ROS. Additionally, the CypA in the nucleus can form DNA degradation complexes with AIF and γ-H2AX, which also promote the execution of chromatinolysis. Thus, we demonstrate that CypA contributes to shikonin-induced glioma cell necroptosis and promotion of chromatinolysis.

Topics: Apoptosis; Apoptosis Inducing Factor; Cyclophilin A; Glioma; Humans; Naphthoquinones; Necroptosis; Receptor-Interacting Protein Serine-Threonine Kinases

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Shikonin plays protective roles in age-associated diseases. Therefore, we investigate the biological functions of shikonin and its mechanisms involved in PD pathogenesis. The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to mimic PD-like conditions in animal models. The learning and memory capacities were assessed by Morris water-maze test, pole test, locomotor activity test and rotarod test. Neuroinflammation was determined by measuring the levels of tumour necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The quantification of superoxide dismutase, malondialdehyde and glutathione in substantia nigra was performed to estimate oxidative damage. Histopathologic changes were examined by haematoxylin and eosin staining. Immunofluorescence staining was conducted to determine the activation of astrocytes, tyrosine hydroxylase (TH)-positive neurons, and nuclear translocation of p65. Immunohistochemistry was performed to evaluate dopamine transporter (DAT)-positive neurons. Protein levels were measured by western blotting. Shikonin alleviates the cognitive and behavioural impairments. The death of dopaminergic neurons in nigra was attenuated by shikonin. The MPTP-induced neuroinflammation and oxidative stress in substantia nigra were alleviated by shikonin administration. Shikonin ameliorated the neuronal damage in nigra and inhibited the activation of astrocyte. Shikonin modulated the protein kinase B (Akt)/extracellular regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/nuclear factor κB (NF-κB) pathways. Shikonin ameliorates dopaminergic neuronal apoptosis by inhibiting oxidative stress and neuroinflammation via the Akt/ERK/JNK/NF-κB pathways in PD. The study has several limitations. First, in a previous study, levels of phosphorylated ERK were increased by MPTP. In our current study, we observed decreased p-ERK in nigra following MPTP treatment. Therefore, further investigation in the mechanisms of shikonin against PD progression is required. Second, the biological functions of shikonin need more exploration, including mitochondrial function and autophagy. Moreover, specific molecular targets for shikonin remain uncertain.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Cyclooxygenase 2; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Glutathione; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Malondialdehyde; Mice; Mice, Inbred C57BL; Naphthoquinones; Neuroinflammatory Diseases; Neurotoxins; NF-kappa B; Nitric Oxide Synthase Type II; Oxidative Stress; Parkinson Disease; Proto-Oncogene Proteins c-akt; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase

Shikonin is well known for its anti-inflammatory activity in cardiovascular diseases. However, the application of shikonin is limited by its low water solubility and poor bioavailability. Methoxy poly (ethylene glycol)-b-poly (ε-caprolactone) (MPEG-PCL) is considered a promising delivery system for hydrophobic drugs. Therefore, in this study, we prepared shikonin-loaded MPEG-PCL micelles and investigated their effect on endothelial-to-mesenchymal transition (EndMT) induced by inflammatory cytokines.. Shikonin was encapsulated in MPEG-PCL micelles using an anti-solvent method and the physiochemical characteristics of the micelles (particle size, zeta potential, morphology, critical micelle concentration (CMC), drug loading and encapsulation efficiency) were investigated. Cellular uptake of micelles in human umbilical vein endothelial cells (HUVECs) was evaluated using fluorescence microscopy. In vitro EndMT inhibition was explored in HUVECs by quantitative real-time PCR analysis.. Shikonin-loaded MPEG-PCL micelles significantly improved the EndMT-inhibiting effect of the free shikonin. MPEG-PCL is suitable for use more generally as a lipophilic drug carrier.

Topics: Anti-Inflammatory Agents; Drug Carriers; Endothelial Cells; Humans; Micelles; Naphthoquinones; Polyesters; Polyethylene Glycols; Tumor Necrosis Factor-alpha; Water

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.

Topics: Acetates; Cyclopentanes; Lithospermum; Mevalonic Acid; Naphthoquinones; Oxylipins; Pharmaceutical Preparations; Plant Growth Regulators; Salicylic Acid

Shikonin is a naphthoquinone compound extracted from Chinese comfrey for treating cancer. However, there are few reports on its research on vertebrate tissue regeneration. Zebrafish is an ideal model for studying organ regeneration. In this study, we found that 3-dpf of zebrafish larvae exposed to shikonin at concentrations of 0.2, 0.3, and 0.4 mg/L showed increasingly inhibited regeneration of the tail fin. Immunohistochemical staining showed that shikonin exposure from 6 to 12 hpa increased the number of apoptotic cells in the caudal fin wound of larvae and decreased the number of proliferating cells. Shikonin exposure was found to up-regulate oxidative stress, increase ROS levels, and reduce neutrophil recruitment in the early stage of wound repair. Moreover, shikonin exposure caused disordered expression of fin regeneration blastemal-related genes. The use of astaxanthin to down-regulate oxidative stress was found to significantly reduce the inhibition of caudal fin regeneration. Mixed exposure of AMPK inhibitors or fullerenes (C60) with shikonin also showed the similar rescue effect. Collectively, our study showed that shikonin inhibited fin regeneration in zebrafish larvae by the upregulation of oxidative stress level and AMPK signaling pathway. This research provides valuable information on the mechanism of action of shikonin for its safe application.

Topics: AMP-Activated Protein Kinases; Animals; Fullerenes; Larva; Naphthoquinones; Reactive Oxygen Species; Zebrafish

Shikonin is the main component of the traditional Chinese medicine comfrey, which can inhibit the activity of PKM2 by regulating glycolysis and ATP production. Rheumatoid arthritis synovial cells (RA-FLSs) have been reported to increase glycolytic activity and have other similar hallmarks of metabolic activity. In this study, we investigated the effects of shikonin on glycolysis, mitochondrial function, and cell death in RA-FLSs. The results showed that shikonin induced apoptosis and autophagy in RA-FLSs by activating the production of reactive oxygen species (ROS) and inhibiting intracellular ATP levels, glycolysis-related proteins, and the PI3K-AKT-mTOR signaling pathway. Shikonin can significantly reduce the expression of apoptosis-related proteins, paw swelling in rat arthritic tissues, and the levels of inflammatory factors in peripheral blood, such as TNF-α, IL-6, IL-8, IL-10, IL-17A, and IL-1β while showing less toxicity to the liver and kidney.

Topics: Adenosine Triphosphate; Animals; Anti-Inflammatory Agents; Apoptosis; Arthritis, Rheumatoid; Autophagy; Cell Line; Disease Models, Animal; Energy Metabolism; Humans; Interleukins; Male; Naphthoquinones; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Synoviocytes; TOR Serine-Threonine Kinases

Choroidal neovascularization (CNV), a feature of neovasular age-related macular degeneration (AMD), acts as a leading cause of vision loss in the elderly. Shikonin (SHI), a natural bioactive compound extracted from Chinese herb radix arnebiae, exerts anti-inflammatory and anti-angiogenic roles and also acts as a potential pyruvate kinase M2 (PKM2) inhibitor in macrophages. The major immune cells macrophages infiltrate the CNV lesions, where the production of pro-angiognic cytokines from macrophage facilitates the development of CNV. PKM2 contributes to the neovascular diseases. In this study, we found that SHI oral gavage alleviated the leakage, area and volume of mouse laser-induced CNV lesion and inhibited macrophage infiltration without ocular cytotoxicity. Moreover, SHI inhibited the secretion of pro-angiogenic cytokine, including basic fibroblast growth factor (FGF2), insulin-like growth factor-1 (IGF1), chemokine (C-C motif) ligand 2 (CCL2), placental growth factor and vascular endothelial growth factor (VEGF), from primary human macrophages by down-regulating PKM2/STAT3/CD163 pathway, indicating a novel potential therapy strategy for CNV.

Topics: Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blotting, Western; Cells, Cultured; Choroidal Neovascularization; Chromatography, High Pressure Liquid; Coloring Agents; Cytokines; Disease Models, Animal; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Fluorescein Angiography; Humans; In Situ Nick-End Labeling; Indocyanine Green; Macrophages; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; Phosphorylation; Pyruvate Kinase; Receptors, Cell Surface; STAT3 Transcription Factor

Overexpressed survivin is associated with worse survival of several types of human tumors. In this study, the antitumor activity of shikonin in non-small-cell lung cancer (NSCLC) by regulating survivin pathway was investigated. Results showed that shikonin inhibited the NSCLC H1299 cell proliferation in a dose-dependent manner. Moreover, shikonin fits well with survivin by molecular docking. Shikonin also inhibited the mRNA expression and protein level of survivin in H1299 cells. Shikonin arrested H1299 cell cycle at the G0/G1 phase by regulating CDK/cyclin family members. In addition, shikonin regulated the expression of X-linked inhibitor of apoptosis- (XIAP-) mediated caspases 3 and 9, thus leading to the damage of mitochondrial membrane potential and induction of H1299 cell apoptosis. Overall, shikonin inhibited H1299 cell growth by inducing apoptosis and blocking the cell cycle. The underlying mechanism involves targeting survivin, which subsequently regulates the protein expression of XIAP/caspase 3/9, CDK2/4, and cyclin E/D1. Thus, shikonin, a survivin inhibitor, is a promising therapeutic strategy in NSCLC treatment.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Naphthoquinones; Signal Transduction; Survivin

Triple-negative breast cancer (TNBC) is heterogeneous disease with a poor prognosis. It is therefore important to explore novel therapeutic agents to improve the clinical efficacy for TNBC. The inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is always overexpressed in many types of tumors, including TNBC and regarded as a potential target for cancer therapy. Through screening a library of natural products, we identified shikonin, a natural bioactive component of Lithospermum erythrorhizon, is a novel and selective IMPDH2 inhibitor. Enzymatic analysis using Lineweaver-Burk plot indicates that shikonin is a competitive inhibitor of IMPDH2. The interaction between shikonin and IMDPH2 was further investigated by thermal shift assay, fluorescence quenching, and molecular docking simulation. Shikonin treatment effectively inhibits the growth of human TNBC cell line MDA-MB-231, and murine TNBC cell line, 4T1 in a dose-dependent manner, which is impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. Most importantly, IMPDH2 knockdown significantly reduced cell proliferation and conferred resistance to shikonin in TNBC. Collectively, our findings showed the natural product shikonin as a selective inhibitor of IMPDH2 with anti-TNBC activity, impelling its further study in clinical trials.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Female; Gene Knockdown Techniques; Humans; IMP Dehydrogenase; Lithospermum; Mice; Molecular Docking Simulation; Naphthoquinones; Triple Negative Breast Neoplasms

A novel intelligent pH-responsive color indicator was prepared by adsorbing a natural naphthoquinone pigment, shikonin, onto cellulose paper. FTIR results indicated that shikonin was crosslinked with the cellulose of the indicator paper. The addition of shikonin increased antioxidant activity, thermal stability, and water resistance properties of the paper. The indicator changed the color from red to dark blue, depending on the pH of buffer solutions. Also, the indicator showed high stability after 4 months of storage and maintained high sensitivity to pH changes. This indicator was used to monitor fish and pork freshness during storage at room temperature, and the results showed a high correlation between the color change of the indicator and the pH change of the sample. The shikonin-adsorbed indicator with stable and sensitive color change depending on pH can be used in the intelligent food packaging applications to monitor the quality of packaged food in real-time.

Topics: Animals; Cellulose; Colorimetry; Fish Products; Food Packaging; Hydrogen-Ion Concentration; Naphthoquinones; Red Meat; Swine

In this study, besides isovaleryl shikonin, another shikonin derivative, tigloylshikonin, was also isolated from the roots of Onosma hookeri Clarke. var. longiforum Duthie as a main naphthoquinone constituent for the first time. Then optimization of the ultrasonic-assisted extraction was done by Box-Behnken design-response surface methodology on the basis of single-factor experiments. The optimized conditions were 72% (v/v) ethanol and the material to solution ratio was 1:37(g/mL) at 52 °C for 77 min. Under these conditions, the extraction yield of ethanol extract was 36.74 ± 0.32%, the contents of isovaleryl shikonin and tigloylshikonin reached 0.094 ± 0.003% and 0.223 ± 0.006%, respectively. Notably, in that optimized condition, the yield of isovaleryl shikonin increased by approximately 7.64-fold than the previous report. In the in vitro antioxidant activity assay, the optimal ethanol extract exhibited similar 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity as butylated hydroxy toluene (BHT), but slightly weaker 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) scavenging activity and total antioxidant capacity than that of BHT. However, the active polar fraction, the ethyl acetate fraction, which is enriched with naphthoquinone constituents, performs as a better antioxidant agent than BHT. Therefore, both of them could be considered as a naturally sourced antioxidants compared to commercially available synthetic drugs. PRACTICAL APPLICATION: Onosma hookeri Clarke. var. longiforum Duthie, a traditional Chinese medicine and food item, has been in use since a long time. A systematic determination of the main naphthoquinones, and antioxidant capacity of the naphthoquinones-enriched ethanol extract and different polar fractions, was carried out in the present study. The results may provide theoretical basis for the claim that naphthoquinones-enriched ethanol extract and ethyl acetate fraction from the roots of Onosma hookeri Clarke. var. longiforum Duthie could be used as potential natural antioxidants in the pharmaceutical, food, and cosmetic industries.

Topics: Antioxidants; Boraginaceae; Free Radical Scavengers; Naphthoquinones; Pentanoic Acids; Plant Extracts; Plant Roots; Ultrasonics

Traditional vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitors can manage angiogenesis; however, severe toxicity and resistance limit their long-term applications in clinical therapy. Shikonin (SHK) and its derivatives could be promising to inhibit the VEGFR-2 mediated angiogenesis, as they are reported to bind in the catalytic kinase domain with low affinity. However, the detailed molecular insights and binding dynamics of these natural inhibitors are unknown, which is crucial for potential SHK based lead design. Therefore, the present study employed molecular modeling and simulations techniques to get insight into the binding behaviors of SHK and its two derivates, β-hydroxyisovalerylshikonin (β-HIVS) and acetylshikonin (ACS). Here the intermolecular interactions between protein and ligands were studied by induced fit docking approach, which were further evaluated by treating QM/MM (quantum mechanics/molecular mechanics) and molecular dynamics (MD) simulation. The result showed that the naphthazarin ring of the SHK derivates is vital for strong binding to the catalytic domain; however, the binding stability can be modulated by the side chain modification. Because of having electrostatic potential, this ring makes essential interactions with the DFG (Asp1046 and Phe1047) motif and also allows interacting with the allosteric binding site. Taken together, the studies will advance our knowledge and scope for the development of new selective VEGFR-2 inhibitors based on SHK and its analogs.

Topics: Binding Sites; Density Functional Theory; Humans; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Naphthoquinones; Protein Kinase Inhibitors; Static Electricity; Vascular Endothelial Growth Factor Receptor-2

In tumor cells, shikonin treatment has been reported to inhibit glycolysis by suppressing the activity of pyruvate kinase M2 (PKM2) and to induce apoptosis by increasing reactive oxygen species (ROS) production. However, hepatocellular carcinoma (HCC) shows variable sensitivity to shikonin treatment, and the mechanism for these differences remains unclear. We evaluated the effects of shikonin on metabolic and oxidative pathways in sensitive and refractory HCC cell lines to identify mechanisms of differential sensitivity.. The sensitivity to shikonin treatment was significantly higher for HepG2 cells than for HCCLM3 cells, with less dramatic effects in HCCLM3 cells on apoptosis, ROS, and oxidative phosphorylation. Shikonin up-regulated mitochondrial biogenesis to increase mitochondrial oxidative phosphorylation in HepG2 cells, but displayed the opposite trend in HCCLM3 cells. Mechanistically, shikonin promoted nuclear expression of PKM2 and HIF1α in HCCLM3 cells, with upregulation of glycolysis-related gene transcription and glycolysis.. These results suggest that PKM2 rewires glucose metabolism, which explains the differential sensitivity to shikonin-induced apoptosis in HCC cells. Our findings elucidate mechanisms for differential responses to shikonin, provide potential biomarkers, and indicate a theoretical basis for targeting glycolytic enzymes in refractory HCC.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma, Hepatocellular; Carrier Proteins; Dose-Response Relationship, Drug; Glucose; Glycolysis; Hep G2 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Membrane Proteins; Naphthoquinones; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Shikonin is the major active component in

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Bronchoalveolar Lavage Fluid; Caspase 3; Cytokines; Disease Models, Animal; DNA Fragmentation; Inflammation; Lipopolysaccharides; Male; Mice, Inbred C57BL; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Neutrophil Infiltration; Neutrophils; Poly(ADP-ribose) Polymerases; Teichoic Acids; Tumor Suppressor Protein p53

Topics: Cell Differentiation; Cells, Cultured; Dental Pulp; Extracellular Matrix Proteins; Hyaluronic Acid; Naphthoquinones; Odontoblasts; Phosphoproteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Stem Cells; TOR Serine-Threonine Kinases

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors. In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model. We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4',6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways). Further, the anticancer effect in vivo was confirmed through a xenograft model. Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner. In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis. Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins. Additionally, shikonin was overexpressed in MAPK pathways. Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, pathologic changes were not observed in the liver and kidney of mice. Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; In Situ Nick-End Labeling; MAP Kinase Signaling System; Melanoma; Mice; Naphthoquinones; Neoplasm Proteins; Xenograft Model Antitumor Assays

Shikonin, a naphthoquinone compound extracted from the root of Lithospermum erythrorhizon, has been extensively studied for its antitumor activity. However, the systematic pathways involved in Shikonin intervention in human colon cancer has not yet clearly defined.. This study was to evaluate the cytotoxic effects of Shikonin in colon cancer, as well as investigate the potential biomarkers from a global perspective and the possible antitumor mechanisms involved.. In this work, cell viability, cell cycle and cell apoptosis in human colon cancer cells were assessed to evaluate the antitumor activity of Shikonin. Transcriptomics and metabolomics were integrated to provide the perturbed pathways and explore the potential mechanisms. The crucial proteins and genes involved were further validated by immunohistochemistry and real-time quantitative PCR.. Shikonin revealed a remarkable antitumor potency in colon cancer. Cell cycle was significantly arrested at the S phase as well as apoptosis was induced in SW480 cell line. Furthermore, a total of 1642 differentially expressed genes and 40 metabolites were detected after Shikonin intervention. The integrated analysis suggested that the antitumor effect was mainly attributed to purine metabolism, arginine biosynthesis, pyrimidine metabolism, urea cycle and metabolism of amino acids. The up-regulated expression of proteins vital for arginine biosynthesis was subsequently validated by immunohistochemistry in xenograft mice. Notably, supplemental dNTPs and arginine could significantly reverse the cytotoxic effect induced by Shikonin and the genes participating in purine metabolism and arginine biosynthesis were further determined by RT-qPCR.. Our findings provide a systematic perspective in the therapeutic effect of Shikonin which might lay a foundation for further research on Shikonin in colon cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Humans; Lithospermum; Metabolomics; Mice; Naphthoquinones; Plant Roots; Xenograft Model Antitumor Assays

Cancer immunotherapy is becoming an important option for malignant tumors treatment. Unfortunately, lacking intratumoral infiltration of cytotoxic T lymphocytes (CTLs) and immunosuppressive tumor microenvironment (ITM) remian primary barriers that immensely hamper its further clinical application. For boosting immune response and rebuilding the ITM, valid hybrid micelles (SK/siIDO1-HMs) delivering shikonin (SK) and IDO-1 knockdown siRNA (siIDO1) were conducted. SK/siIDO1-HMs had sufficient circulation time, favorable intratumoral accumulation and rapidly release in the cytoplasm. Importantly, SK was demonstrated to significantly elicit intratumoral accumulation of CTLs through inducing immunogenic cell death (ICD) of tumor cells. Moreover, siIDO1 downregulated the IDO-1-caused immunosuppression and restrained regulatory T lymphocytes (Tregs). In summary, SK/siIDO1-HMs displayed a remarkable potential for tumor therapy via triggering the ICD and moderating the IDO-1-triggered immunosuppression.

Topics: Immunotherapy; Micelles; Naphthoquinones; RNA, Small Interfering; Tumor Microenvironment

Cotton is an important renewable biopolymer with extensive applications in various fields including textiles. In the current study a soy protein (SP) crosslinked cotton fabric (SPCCF) was prepared through the reaction of carboxyl cotton fabric with soy protein without using crosslinking agents. FTIR analysis of SPCCF samples indicated that carboxyl groups in oxycellulose fabric have reacted with amino groups of SP to give the corresponding C-N bond, that was also reconfirmed by XPS spectra and TGA/DTG analyses of the grafted fabrics. The resulting SPCCF fabrics acquired under the optimized conditions exhibited the improved tensile strength and capillary effect as compared to the oxidized cotton fabric. The ungrafted and grafted fabrics were further evaluated for dyeing property, as a result, the SPCCF fabrics showed markedly improved colour strength when dyed with acid dyes. The fastness properties of dyeability for the dyed SPCCF fabrics were also good compared with that of ungrafted fabrics by dyeing. Shikonin as a kind of Chinese medicine was found to immobilize on the SPCCF fabric through treatment with shikonin aqueous solution, such fabric displayed effective antibacterial activities against both gram-positive and gram-negative bacteria with durability of 30 washes. These results suggest that the SPCCF can be suitable for medical protective textiles by immobilizing drugs.

Topics: Anti-Bacterial Agents; Cellulose, Oxidized; Color; Coloring Agents; Cotton Fiber; Escherichia coli; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Naphthoquinones; Oxygen; Photoelectron Spectroscopy; Protein Conformation; Soybean Proteins; Spectroscopy, Fourier Transform Infrared; Staphylococcus aureus; Temperature; Textiles; Thermogravimetry; Time Factors

Pancreatic cancer (PC) is a highly fatal malignancy with few effective therapies currently available. Recent studies have shown that PD-L1 inhibitors could be potential therapeutic targets for the treatment of PC. The present study aims to investigate the effect of Shikonin on immune evasion in PC with the involvement of the PD-L1 degradation.. Initially, the expression patterns of PD-L1 and NF-κB in PC were predicted in-silico using the GEPIA database, and were subsequently validated using PC tissues. Thereafter, the correlation of NF-κB with STAT3, CSN5 and PD-L1 was examined. PC cells were treated with Shikonin, NF-κB inhibitor, STAT3 activator, and CSN5 overexpression plasmid to investigate effects on PD-L1 glycosylation and immune evasion in PC. Finally, in vivo tumor formation was induced in C57BL/6J mice, in order to verify the in vitro results.. PD-L1, NF-κB, NF-κB p65, STAT3, and CSN5 were highly expressed in PC samples, and NF-κB was positively correlated with STAT3/CSN5/PD-L1. Inhibition of NF-κB decreased PD-L1 glycosylation and increased PD-L1 degradation, whereas activated STAT3 and overexpressed CSN5 reversed these trends. Shikonin blocked immune evasion in PC, and lowered the expression of PD-L1, NF-κB, NF-κB p65, STAT3 and CSN5 in vivo and in vitro.. The findings indicated Shikonin inhibited immune evasion in PC by inhibiting PD-L1 glycosylation and activating the NF-κB/STAT3 and NF-κB/CSN5 signaling pathways. These effects of Shikonin on PC cells may bear important potential therapeutic implications for the treatment of PC.

Topics: Animals; Antineoplastic Agents; B7-H1 Antigen; Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; COP9 Signalosome Complex; Flow Cytometry; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; NF-kappa B; Pancreatic Neoplasms; Peptide Hydrolases; Signal Transduction; STAT3 Transcription Factor; Tumor Escape; Xenograft Model Antitumor Assays

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

Antitumor effects of shikonins on chronic lymphocytic leukemia (CLL) and B-cell prolymphocytic leukemia (B-PLL) are mostly unexplored. The antitumor activity of shikonins, isolated from

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Boraginaceae; Cell Line; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Leukemia; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Naphthoquinones; Phosphoproteins; STAT3 Transcription Factor

Topics: Anilides; Antineoplastic Agents; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Esters; Humans; Molecular Structure; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Structure-Activity Relationship; TOR Serine-Threonine Kinases

Natural colorants (anthocyanin and shikonin) were blended in different ratios (3:1 and 1:3) and used for the preparation of carboxymethyl cellulose (CMC)/agar-based functional halochromic films. The colorants were compatible with the polymer matrix and evenly spread over the polymer matrix. The addition of colorants slightly improved the mechanical strength and significantly improved the water vapor barrier properties of CMC/agar-based films without altering the thermal stability. The color indicator film exhibited excellent UV- barrier properties without substantially reducing the transparency. It also showed distinct pH-responsive color-changing properties in the pH range of 2-12, showing excellent acid and base gas sensing properties. The shikonin-added film showed potent antimicrobial activity against food-borne pathogenic bacteria, and the color indicator films exhibited intense antioxidant activities. The CMC/agar-based color indicator films with improved physical and functional properties are likely to be used in active and intelligent food packaging applications.

Topics: Agar; Anthocyanins; Anti-Infective Agents; Carboxymethylcellulose Sodium; Naphthoquinones

Shikonin and its derivatives are the main components of traditional Chinese medicine, Zicao. The pharmacological potential of shikonin and its derivatives have been extensively studied. Yet, less is known about the microbial assemblages associated with shikonin producing Borage plants. We studied microbial profiles of two Borage species, Echium plantagineum (EP) and Lithospermum erythrorhizon (LE), to identify the dynamics of microbial colonization pattern within three rhizo-compatments and two distinct soil types. Results of α and β-diversity via PacBio sequencing revealed significantly higher microbial richness and diversity in the natural soil along with a decreasing microbial gradient across rhizosphere to endosphere. Our results displayed genotype and soil type-dependent fine-tuning of microbial profiles. The host plant was found to exert effects on the physical and chemical properties of soil, resulting in reproducibly different micro-biota. Analysis of differentially abundant microbial OTUs displayed Planctomycetes and Bacteroidetes to be specifically enriched in EP and LE rhizosphere while endosphere was mostly prevailed by Cyanobacteria. Network analysis to unfold co-existing microbial species displayed different types of positive and negative interactions within different communities. The data provided here will help to identify microbes associated with different rhizo-compartments of potential host plants. In the future, this might be helpful for manipulating the keystone microbes for ecosystem functioning.

Topics: Bacteria; Borago; DNA, Bacterial; DNA, Ribosomal; Hydrogen-Ion Concentration; Naphthoquinones; Phylogeny; Plant Leaves; Plant Roots; Rhizosphere; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology

Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 8; Cell Line, Tumor; Cell Proliferation; Connexins; Gap Junction beta-1 Protein; Gene Knockdown Techniques; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Necroptosis; Nuclear Receptor Coactivator 1; Phosphorylation; Signal Transduction; Transfection; Tumor Burden

Chemo-immunotherapy based on immunogenic cell death (ICD) is a promising strategy for cancer therapy. However, the effective ICD requires a high dosage of ICD stimulus, which could be associated to a dose-dependent toxicity. Therefore, in this study, a liposome remote-loaded with shikonin (a potent ICD stimulus) was developed, with the ability to effectively induce ICD at high dosage in vivo. However, a hepatotoxic effect was observed. To circumvent this problem, shikonin was combined with the anthracycline mitoxantrone or doxorubicin to develop co-loaded liposomes inducing a synergistic ICD effect and cytotoxicity to tumor cells. Cytotoxicity and uptake experiment in vitro were performed to analyze the optimal synergistic ratio of shikonin and anthracyclines based on a "formulated strategy". Interestingly, copper mediated co-loaded liposomes resulted in a pH and GSH dual-responsive release property. More importantly, pharmacokinetics and tumor biodistribution studies revealed an outstanding capacity of ratiometric delivery of dual drugs. Thus, the dual-loaded liposome enhanced the antitumor effect by the stimulation of a robust immune response at lower doses of the drugs with a higher safety compared to single-loaded liposomes. Summarized, the current work provided a reference for a rational design and development of liposomal co-delivery system of drugs and ICD-induced chemo-immunotherapy, and established a potential clinical application of shikonin-based drug combinations as a new chemo-immunotherapeutic strategy for cancer treatment.

Topics: Anthracyclines; Cell Line, Tumor; Doxorubicin; Humans; Immunogenetics; Immunogenic Cell Death; Immunotherapy; Liposomes; Naphthoquinones; Tissue Distribution

Pneumonia is an inflammatory disease induced by infection with different pathogens. Currently, multiple preclinical studies have revealed that shikonin, a natural naphthoquinone, can mitigate lipopolysaccharide (LPS)-induced inflammation, but its underlying mechanism in pneumonia remains unknown. This research was designed to explore the function and regulatory mechanism of shikonin in LPS-induced cell injury and inflammation in WI-38 cells. In-vitro model of pneumonia was constructed by treating WI-38 cells with LPS. Expression of miR-489-3p and MAP2K1 was tested by RT-qPCR and (or) Western blot analysis. Cell viability was examined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay. The productions of pro-inflammatory cytokines were determined by enzyme-linked immunosorbent assays. Cell apoptosis was detected by Western blot and flow cytometry analysis. In the current study, LPS induced WI-38 cell damage by inhibiting cell viability and promoting cell apoptosis and inflammation. Shikonin ameliorated LPS-induced cell injury and elevated miR-489-3p expression. LPS-induced inflammatory injury was further mitigated by upregulation of miR-489-3p. In addition, MAP2K1, the target of miR-489-3p, was upregulated by LPS. Furthermore, upregulation of MAP2K1 reversed the influence of shikonin and miR-489-3p mimics on LPS-induced cell injury and inflammation. This study revealed that shikonin protected WI-38 cells against LPS-induced cell injury and inflammatory response by regulating the miR-489-3p/MAP2K1 axis, thus affecting the progression of pneumonia.

Topics: Apoptosis; Lipopolysaccharides; MicroRNAs; Naphthoquinones

Lung cancer is one of the most lethal diseases and therefore poses a significant threat to human health. The Warburg effect, which is the observation that cancer cells predominately produce energy through glycolysis, even under aerobic conditions, is a hallmark of cancer. 6‑phosphofructo‑2‑kinase/fructose‑2,6‑biphosphatase 2 (PFKFB) is an important regulator of glycolysis. Shikonin is a Traditional Chinese herbal medicine, which has been reported to exert antitumor effects. The present study aimed to investigate the anticancer activity of shikonin in lung cancer. Cell Counting Kit‑8 (CCK‑8) and colony formation assays were used to analyze proliferation in A549 and H446 cells. Wound healing and Transwell assays were used to measure migration and invasion in A549 and H446 cells. Cell apoptosis was analyzed using flow cytometry. Lactate levels, glucose uptake and cellular ATP levels were measured using their corresponding commercial kits. Western blotting was performed to analyze the protein expression levels of key enzymes involved in aerobic glucose metabolism. Reverse transcription‑quantitative PCR was used to analyze the mRNA expression levels of PFKFB2. The results of the present study revealed that PFKFB2 expression levels were significantly upregulated in NSCLC tissues. Shikonin treatment decreased the proliferation, migration, invasion, glucose uptake, lactate levels, ATP levels and PFKFB2 expression levels and increased apoptosis in lung cancer cells in a dose‑dependent manner. The overexpression of PFKFB2 increased the proliferation, migration, glucose uptake, lactate levels and ATP levels in lung cancer cells, while the knockdown of PFKFB2 expression exerted the opposite effects. Moreover, there were no significant differences in lung cancer cell migration, apoptosis, glucose uptake, lactate levels and ATP levels between cells with knocked down PFKFB2 expression or treated with shikonin and the knockdown of PFKFB2 in cells treated with shikonin. In conclusion, the results of the present study revealed that shikonin inhibited the Warburg effect and exerted antitumor activity in lung cancer cells, which was associated with the downregulation of PFKFB2 expression.

Topics: Aged; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Lung Neoplasms; Male; Middle Aged; Naphthoquinones; Phosphofructokinase-2; Up-Regulation; Warburg Effect, Oncologic

Topics: Allosteric Regulation; Enzyme Inhibitors; Humans; Hypoglycemic Agents; Molecular Docking Simulation; Naphthoquinones; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Structure-Activity Relationship

To investigate the effects of shikonin on CD36 expression and phagocytic ability of microglia, and its protective effect on neurons and the possible mechanism within.. The effects of shikonin on CD36 expression and phagocytic ability of microglia were detected by Western blot method, and cerebral haemorrhage was isolated by flow cytometry in the experiment. The protective effect of neurons was observed through neuron-microglia co-culture technique. Meanwhile, the effect of hydrogen peroxide on the expression of catalase was detected, and the concentration of hydrogen peroxide was measured in the isolated cerebral haemorrhage model. The t test was used to compare data between 2 groups, and one-way ANOVA was applied to multiple sets of data.. Compared with the control group, the CD36 expression and phagocytic ability of microglia was increased by shikonin in the isolated cerebral haemorrhage model, while inflammatory factors such as tumour necrosis factor a (TNF-a) and interleukin 1b (IL-1b) attenuated the effects of the drug. The amount of neuron apoptosis/necrosis was significantly reduced by the drug, while the expression of catalase in microglia was increased, but the secretion of hydrogen peroxide was decreased in the neuron-microglia co-culture system.. Shikonin can enhance the CD36 expression and the ability to phagocytose erythrocyte of microglia. Simultaneously, shikonin performs protective effects on neuronal cells and promotes the absorption of haematoma. Therefore, shikonin is probably an innovative medicine to treat cerebral haemorrhage.

Topics: CD36 Antigens; Cerebral Hemorrhage; Humans; Microglia; Naphthoquinones

To investigate the effects and potential mechanism of action of shikonin (SHK) on the development of ovarian follicles and female germline stem cells (FGSCs).. Female Kunming adult mice were administered SHK (0, 20 and 50 mg/kg) by oral gavage. Cultures of FGSCs were treated with SHK 32 μmol/l for 24 h. The ovarian index in mouse ovaries was calculated. Numbers of primordial, primary and atretic follicles were counted. Germline stem cell markers and apoptosis were examined. Levels of glutathione (GSH), superoxide dismutase (SOD) and reactive oxygen species (ROS) were measured.. Both doses of SHK significantly decreased the ovarian index, the numbers of primordial follicles, primary follicles and antral follicles in mice. SHK significantly increased the numbers of atretic follicles and atretic corpora lutea. SHK promoted apoptosis. These current results suggested that follicular development and FGSCs were suppressed by SHK through the induction of apoptosis and oxidative stress might be involved in this pathological process.

Topics: Animals; Apoptosis; Female; Mice; Naphthoquinones; Oogonial Stem Cells; Ovarian Follicle

Pyruvate kinase isozyme M2 (PKM2) was observed to be overexpressed and play a key role in cell growth and cancer cells' metabolism. During the past years, phytochemicals have been developed as new treatment options for chemoprevention and cancer therapy. Natural resources, like shikonin (naphthoquinone) and its derivatives, have emerged to be high potential therapeutics in cancer treatment.. Our study aimed to design novel anti-tumour agents (PKM2 inhibitors) focusing on the shikonin scaffold with a better activity using computational methods. We applied a three-dimensional quantitative structure-activity relationship (3D-QSAR) approach using Field-based QSAR.. The Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) were performed on a series of forty shikonin derivatives, including shikonin, to develop robust models and rationalize the PKM2 inhibitory activity profile by building a correlation between structural features and activity.. These predictive computational models will further help the design and synthesis of potent PKM2 inhibitors and their fast biological assessment at a low cost.

Topics: Enzyme Inhibitors; Isoenzymes; Naphthoquinones; Pyruvate Kinase; Quantitative Structure-Activity Relationship

Ambrisentan is an oral endothelin-receptor antagonist (ERA). However, there is no report on the interaction between ambrisentan and shikonin.. To investigate the effect of shikonin on ambrisentan metabolism. The UPLC-MS/MS method was shown to be accurate, precise and reliable, and was successfully applied to the herb-drug interaction study of ambrisentan with shikonin. When co-administrated with 20 mg/kg shikonin, the. Our study indicated that shikonin could inhibit ambrisentan metabolism. Further studies need to be carried out to verify whether similar interaction truly apply in humans and whether this interaction has clinical significance.

Topics: Animals; Area Under Curve; Chromatography, High Pressure Liquid; Herb-Drug Interactions; Humans; Male; Microsomes, Liver; Naphthoquinones; Phenylpropionates; Pyridazines; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Tandem Mass Spectrometry

Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be ∼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contigN50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon's roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT's functional differentiation in evolution.

Topics: Acyltransferases; Lithospermum; Naphthoquinones

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

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

Shikonin (SH) is used as a red pigment for food coloring and cosmetics, and has cytotoxic activity towards cancer cells. However, due to strong toxicity SH has limited potential as an anticancer drug. Acetylshikonin (ASH) is one of the SH derivatives with promising anticancer potential. In present study, we attempted to evaluate and compare the cytotoxicity of SH and ASH towards a normal cell line (V79) and in addition to evaluate their antigenotoxic activity. The evaluation was made with the use of the set of cytotoxicity assays with V79 line and the micronucleus test

Topics: Animals; Anthraquinones; Cell Line; Cricetulus; Cyclophosphamide; DNA Damage; Ethyl Methanesulfonate; Fluoroquinolones; Micronucleus Tests; Naphthoquinones

Shikonin is a natural naphthoquinone component with antioxidant and anti-tumor function and has been used for hepatocellular carcinoma (HCC) treatment. According to the previous study, many herbs can regulate cancer cell progression by targeting specific microRNA (miRNA) (Liu, 2016). However, the underlying pathological mechanism of shikonin in HCC therapy is still unclear. The detection of cell growth and death rate were performed by hemacytometry and trypan blue staining, respectively. The expression of miR-106b and SMAD7 messenger RNA (mRNA) in HCC cells was evaluated by quantitative real-time polymerase chain reaction. Cell proliferation, apoptosis, and migration ability were measured by cell counting kit-8 (CCK-8), flow cytometry, and transwell assay. The expression of proteins E-cadherin, N-cadherin, vimentin, SMAD7, TGF-β1, p-SMAD3, SMAD3, and GAPDH was examined by western blot. The interaction between SMAD7 and miR-106b was assessed by luciferase reporter system. Shikonin inhibited Huh7 and HepG2 cell growth in a dose-dependent manner while induced cell death in a time-dependent manner. In addition, the expression of miR-106b was reduced after shikonin treatment. Moreover, miR-106b attenuated the suppressive effects of shikonin on HCC cell migration and epithelial-mesenchymal transition (EMT). SMAD7 was predicted as a target of miR-106b and the prediction was confirmed by luciferase reporter system. Additionally, we observed that SMAD7 reversed the promotive effects of miR-106b on HCC cell progression and EMT. The subsequent western blot assay revealed that shikonin could modulate SMAD7/TGF-β signaling pathway by targeting miR-106b. In conclusion, Shikonin suppresses cell progression and EMT and accelerates cell death of HCC cells via modulating miR-106b/SMAD7/TGF-β signaling pathway, suggesting shikonin could be an effective agent for HCC treatment.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; Naphthoquinones; Smad7 Protein; Transforming Growth Factor beta1; Tumor Cells, Cultured

Boraginales (the forget-me-not order) is a core group within the lamiids clade. However, until now, no genome from Boraginales has been reported, and published transcriptomes are also rare. Here, we report the first Boraginales species de novo genome (i.e. Echium plantagineum genome) and seven other Boraginales species transcriptomes to probe three issues: (i) Boraginales' phylogenetic position within the lamiids clade; (ii) potential whole genome duplications (WGDs) in Boraginales; and (iii) candidate key enzyme genes in the alkannin/shikonin core pathway. The results showed that: (i) Boraginales was most probably closer to the Solanales/Gentianales clade than the Lamiales clade, at least based on the single-copy orthologous genes from genome/transcriptome data; (ii) after the gamma (γ) event, Boraginaceae (classified into the Boraginales I clade) probably underwent at least two rounds of WGD, whereas Heliotropiaceae and Ehretiaceae (classified into the Boraginales II clade) probably underwent only one round of WGD; and (iii) several candidate key enzyme genes in the alkannin/shikonin core pathway were inferred, e.g. genes corresponding to geranyl cyclase, naphthol hydroxylase and O-acyl transferase.

Topics: Biosynthetic Pathways; Gene Duplication; Gene Expression Profiling; Genome, Plant; Magnoliopsida; Naphthoquinones; Phylogeny; Plant Proteins; Transcriptome

Shikonin is an anti-inflammatory agent extracted from natural herbs. The aim of this study is to explain the treatment effects and mechanism of Shikonin in acute lung injury induced by sepsis. In this study, first, we evaluate different Shikonin concentrations for the anti-inflammation of acute lung injury induced by sepsis in an in vivo study. On the basis of the results, we confirm that 50.0 mg/kg was the best therapeutic Shikonin concentration. As a second step, we discuss the mechanism of Shikonin by a vitro cell experiment. Finaly, we validate that Shikonin has effective treatment effects on acute lung injury via regulation of microRNA-140-5p/toll-like receptor 4 (miRNA-140-5p/TLR4) in the in vivo study. The results of vitro and vivo study showed that Shikonin could improve acute lung injury induced by sepsis. The mechanism might be correlation miRNA-140-5p expression increasing, and regulated targeted gene TLR4, with TLR4 expression depressing, the downstream myeloid differentiation protein 88 and nuclear factor κB proteins expression were suppressed. In conclusion, Shikonin improved sepsis induced lung injury by regulation miRNA-140-5p/TLR4.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Proliferation; Cells, Cultured; Gene Expression Regulation; Male; MicroRNAs; Naphthoquinones; Rats; Sepsis; Specific Pathogen-Free Organisms; Toll-Like Receptor 4

Benzoxepane derivatives were designed and synthesized, and one hit compound emerged as being effective in vitro with low toxicity. In vivo, this hit compound ameliorated both sickness behavior through anti-inflammation in LPS-induced neuroinflammatory mice model and cerebral ischemic injury through anti-neuroinflammation in rats subjected to transient middle cerebral artery occlusion. Target fishing for the hit compound using photoaffinity probes led to identification of PKM2 as the target protein responsible for anti-inflammatory effect of the hit compound. Furthermore, the hit exhibited an anti-neuroinflammatory effect in vitro and in vivo by inhibiting PKM2-mediated glycolysis and NLRP3 activation, indicating PKM2 as a novel target for neuroinflammation and its related brain disorders. This hit compound has a better safety profile compared to shikonin, a reported PKM2 inhibitor, identifying it as a lead compound in targeting PKM2 for the treatment of inflammation-related diseases.

Topics: Animals; Anti-Inflammatory Agents; Dibenzoxepins; Disease Models, Animal; Infarction, Middle Cerebral Artery; Interleukin-1beta; Ischemic Stroke; Lipopolysaccharides; Macrophages; Mice; Microglia; Naphthoquinones; NLR Family, Pyrin Domain-Containing 3 Protein; Pyruvate Kinase; Rats; RAW 264.7 Cells; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Topics: A549 Cells; Apoptosis; Cell Survival; Drug Synergism; Humans; Lung Neoplasms; Metal Nanoparticles; Naphthoquinones; Silver

Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca. PDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca. The PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca. Cutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.

Topics: Adenosine Triphosphate; Calcium; Carcinoma, Pancreatic Ductal; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cytosol; Glycolysis; Humans; Membrane Proteins; Naphthoquinones; Pancreas; Pancreatic Neoplasms; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Shikonin, shikonofuran and their derivatives are the main bioactive components of Zicao, a traditional Chinese medicine prepared with the dried roots of Lithospermum erythrorhizon, Arnebia euchroma or Arnebia guttata. To establish an efficient and sensitive method for studying material basis of Zicao, different scan modes of ultra-high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and UHPLC triple quadrupole linear ion trap mass spectrometry (QTRAP-MS/MS) were incorporated to make full use of the sensitivity of multiple reaction monitoring (MRM) and overcome its disadvantages. A total of 73 shikonins and shikonofurans compounds were detected in Zicao utilizing various scanning modes. Thereafter the characteristic chemical profile for shikonins and shikonofurans was established based on UHPLC-QTRAP-MS/MS, which was subsequently used to study the spectrum-effect relationship by correlating the relative quantity of compounds and the anti-tumor activity. As a result, 27 compounds were screened as the main active components inhibiting HeLa cells by othogonal partial least square (OPLS). Among them, shikonin, acetylshikonin have been reported to inhibit HeLa cells previously, and β, β-dimethylacrylshikonin has been reported to be active component by other method. Those results showed that chemical characteristic profile combined with chemometric methods was efficient and reliable for discovery of material basis in TCM, especially trace active compounds.

Topics: Antineoplastic Agents; Cell Proliferation; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Furans; HeLa Cells; Humans; Least-Squares Analysis; Naphthoquinones; Tandem Mass Spectrometry

Increased glycolysis is involved in the proliferation and migration of vascular smooth muscle cells (VSMCs). Pyruvate kinase isoform M2 (PKM2), a key rate-limiting enzyme in glycolysis, accelerates the proliferation and migration of tumor cells. Although the intracellular mechanisms associated with oxidized low-density lipoprotein (oxLDL)-stimulated VSMC proliferation and migration have been extensively explored, it is still unclear whether oxLDL promotes the proliferation and migration of VSMCs by enhancing PKM2-dependent glycolysis. In the present study, we detected PKM2 expression and pyruvate kinase activity in oxLDL-treated VSMCs and explored the regulation of PKM2 in oxLDL-treated VSMCs and apoE-/- mice. The results showed that PKM2 expression in VSMCs was higher in the intima than in the media in plaques from atherosclerotic rabbits. Moreover, PKM2 level in VSMCs was increased during atherosclerosis progression in apoE-/- mice. Both PKM2 expression and pyruvate kinase activity were found to be upregulated by oxLDL stimulation in VSMCs. Shikonin (SKN), a specific inhibitor of PKM2, was found to inhibit the oxLDL-induced proliferation and migration in VSMCs, in addition to delaying the atherosclerosis progression in apoE-/- mice. More importantly, oxLDL increased glucose uptake, ATP and lactate production, and the extracellular acidification rate in VSMCs, which could be reversed by SKN. Meanwhile, oxygen consumption rate was unchanged after oxLDL stimulation, suggesting that glycolysis is the main contributor to the energy supply in oxLDL-treated VSMCs. Our results suggest that oxLDL induces VSMC proliferation and migration by upregulating PKM2-dependent glycolysis, thereby contributing to the atherosclerosis progression. Thus, targeting PKM2-dependent glycolysis might provide a novel therapeutic approach for the treatment of atherosclerosis.

Topics: Animals; Atherosclerosis; Carrier Proteins; Cell Movement; Cell Proliferation; Cells, Cultured; Gene Knockdown Techniques; Glycolysis; Humans; Lipoproteins, LDL; Male; Membrane Proteins; Mice; Mice, Knockout, ApoE; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthoquinones; Pyruvate Kinase; Rabbits; Rats; Rats, Sprague-Dawley; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Colorectal carcinoma (CRC) is the third most common malignant tumor pathology worldwide. Despite progress in surgical procedures and therapy options, CRC is still a considerable cause of cancer-related mortality. In this study, we tested the antitumor effects of shikonin in CRC and tried to identify its potential mechanism. The potential target, molecular mechanism as well as

Topics: Apoptosis; Autophagy; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Galectin 1; Humans; MAP Kinase Kinase 4; Naphthoquinones; Reactive Oxygen Species; RNA Interference

Geranyl diphosphate (GPP) is the direct precursor of all monoterpenoids and is the prenyl source of many meroterpenoids, such as geranylated coumarins. GPP synthase (GPPS) localized in plastids is responsible for providing the substrate for monoterpene synthases and prenyltransferases for synthesis of aromatic substances that are also present in plastids, but GPPS activity in

Topics: Coumarins; Cytosol; Geranyltranstransferase; Lithospermum; Monoterpenes; Mutagenesis, Site-Directed; Naphthoquinones; Plastids

Exploiting metabolic vulnerabilities of cancer cells with nontoxic, plant derived compounds constitutes a novel strategy for both chemoprevention and treatment. A high-throughput screening approach was used to evaluate a library of natural products to determine the most synergistic combination in precursor-B cell acute lymphoblast leukemia. Dimethylaminoparthenolide and shikonin effectively inhibited proliferation resulting in cell death in primary and immortalized leukemia cells, while having negligible effects on normal cells. Dimethylaminoparthenolide and shikonin have been shown separately to inhibit cell survival and proliferative signaling and activate tumor suppressors and proapoptotic pathways. Untargeted metabolomics and metabolic flux analysis with stable isotopically labeled glucose and glutamine exhibited a global shift in metabolism following treatment. Pathway analysis indicated significant differences in amino acid, antioxidant, tricarboxylic acid cycle, and nucleotide metabolism. Together, dimethylaminoparthenolide and shikonin reduced the shunting of glycolytic intermediates into the pentose phosphate pathway for biosynthetic purposes. Similarly, the incorporation of glutamine and glutamine-derived metabolites into purine and pyrimidine synthesis was inhibited by the combination of dimethylaminoparthenolide and shikonin, effectively impeding biosynthetic pathways critical for leukemia cell survival. This approach demonstrates that a synergistic pair of compounds with malignant cell specificity can effectively target metabolic pathways crucial to leukemia cell proliferation and induce apoptosis.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Child; Citric Acid Cycle; Drug Screening Assays, Antitumor; Drug Synergism; Glucose; Glutamine; Glycolysis; Humans; Metabolic Networks and Pathways; Naphthoquinones; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Sesquiterpenes

Mast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China.. To investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects.. The anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca. Shikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein.. Shikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca

Topics: Anaphylaxis; Animals; Calcium; Cell Degranulation; Cell Line; Chemokines; Cytokines; Humans; Hypersensitivity; Male; Mast Cells; Mice, Inbred C57BL; Naphthoquinones; Nerve Tissue Proteins; p-Methoxy-N-methylphenethylamine; Phospholipase C gamma; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Secretagogues

Topics: Antineoplastic Agents; Apoptosis; Benzofurans; Binding Sites; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Human Umbilical Vein Endothelial Cells; Humans; Molecular Docking Simulation; Naphthoquinones; Protein Binding; Tubulin; Tubulin Modulators

NSCLC is the major type of lung cancer and the survival rates of NSCLC patients remain low. AZD9291 is a third-generation EGFR-TKI and approved to treat NSCLC patients harboring EGFR T790M mutation and common targetable activating EGFR mutations, but it has a limited effect for wtEGFR NSCLC.. The current study investigated whether shikonin could enhance the antitumor effect of AZD9291 in wtEGFR NSCLC cells.. SRB and colony formation assay were used to detect the proliferation of NSCLC cells, propidium iodide staining was performed to detect the apoptosis, ROS was analyzed using DCFH-DA staining, and western blot was used to detect the expression of indicated proteins.. We demonstrated that shikonin, a natural ROS inducer, could enhance the antitumor effect of AZD9291 in wtEGFR NSCLC cells. In addition, shikonin increased AZD9291-induced apoptosis accompanying with the generation of ROS and activation of ER stress. Furthermore, ROS inhibition by NAC or GSH reversed the apoptosis induced by shikonin plus AZD9291, and recovered the ER stress activated by combination treatment, indicating that ROS mediated ER stress played a vital role in this combination therapy. Moreover, shikonin increased the anticancer activity of AZD9291 in primary wtEGFR NSCLC cells through ROS-mediated ER stress.. Our study suggests that combining shikonin with AZD9291 is a promising therapeutic strategy for treating wtEGFR NSCLC patients.

Topics: A549 Cells; Acrylamides; Aniline Compounds; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Synergism; Endoplasmic Reticulum Stress; ErbB Receptors; Humans; Lung Neoplasms; Naphthoquinones; Protein Kinase Inhibitors; Reactive Oxygen Species

Shikonin, the main ingredient of. MTT, wound-healing, transwell assays and flow cytometry experiments were used to measure cell growth, migration, invasion, and cell cycle analysis. Western blot was used to examine protein levels of Snail, Vimentin and E-cadherin. The expression level of miR-183-5p was measured via qRT-PCR. The E-cadherin promoter activity was detected via Secrete-PairTM Dual Luminescence Assay Kit. The transient transfection experiments were used for silencing of E-cadherin and overexpression of Snail genes. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings.. We showed that shikonin inhibited cell viability, migration and invasion, and induced cell cycle arrest in a dose-dependent manner in cervical cancer Hela and C33a cells. Mechanistically, we found that shikonin increased miR-183-5p expression and inhibited expression of transcription factor Snail protein. The mimics of miR-183-5p reduced, while the inhibitors of miR-183-5p reversed shikonin-inhibited Snail protein expression. In addition, shikonin decreased Vimentin, increased E-cadherin protein expressions and E-cadherin promoter activity, the latter was reversed in cells transfected with exogenous Snail overexpression vectors. Moreover, silencing of E-cadherin significantly abolished shikonin-inhibited cervical cancer cell growth. Similar findings were also observed in vivo using one xenograft mouse model.. Our results show that shikonin inhibits EMT through inhibition of Snail and stimulation of miR-183-5p expressions, which resulted in induction of E-cadherin expression. Thus, blockade of EMT could be a novel mechanism underlying the anti-cervical cancer effects of shikonin.

Topics: Animals; Antigens, CD; Antineoplastic Agents, Phytogenic; Cadherins; Cell Line, Tumor; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Lithospermum; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Naphthoquinones; Snail Family Transcription Factors; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

This study is aimed at investigating the effects of shikonin, a pyruvate kinase M2 (PKM2) inhibitor, on the functions of myeloid dendritic cells (mDCs) in a mouse model of severe aplastic anemia (AA) generated by total body irradiation and lymphocyte infusion. Flow cytometry and qPCR were used to determine the proportions of PKM2+ mDCs and other immune indicators in the AA mice. Glucose consumption level, pyruvate generation level, and ATP content were used to determine the level of glycolytic metabolism in the mDCs. The survival rates of AA mice were evaluated after the administration of shikonin or the immunosuppressive agent cyclosporin A. The AA mice displayed pancytopenia, decreased CD4+/CD8+ cell ratio, increased perforin and granzyme levels in CD8+ cells, increased costimulatory CD80 and CD86 expressions, and inadequate regulatory T cell number.

Topics: Anemia, Aplastic; Animals; B7-1 Antigen; B7-2 Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cyclosporine; Dendritic Cells; Flow Cytometry; Immunosuppressive Agents; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Naphthoquinones; Pyruvate Kinase; Reverse Transcriptase Polymerase Chain Reaction

The development of paclitaxel-resistance led to the tumor relapse and treatment failure of non-small cell lung cancer. Shikonin has been demonstrated to show anti-cancer activity in many cancer types. The present study aimed to investigate the anti-cancer activity of shikonin in paclitaxel-resistant non-small cell lung cancer treatment.. MTT, clonogenic assay, apoptotic cell death analysis, western blot, qRT-PCR, gene knockdown and overexpression, xenograft experiment, immunohistochemistry were performed.. Shikonin decreased paclitaxel-resistant NSCLC cell viability and inhibited the growth of xenograft tumor. Shikonin induced apoptotic cell death of paclitaxel-resistant NSCLC cell lines and suppressed the level of NEAT1 and Akt signaling of paclitaxel-resistant NSCLC cell lines and xenograft tumors. Either low dose or high dose of shikonin considerably suppressed the cell growth and induced the cell apoptotic death in NEAT1 knockdown A549/PTX cells, and p-Akt expression was decreased.. Shikonin could be a promising candidate for paclitaxel-resistant NSCLC treatment.

Topics: A549 Cells; Animals; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; Naphthoquinones; Paclitaxel; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; Xenograft Model Antitumor Assays

Postmenopausal osteoporosis results from estrogen withdrawal and is characterized mainly by bone resorption. Shikonin is a bioactive constitute of Chinese traditional herb which plays a role in antimicrobial and antitumor activities. The study was designed to investigate the role of shikonin on postmenopausal osteoporosis and explore its underlying mechanisms.. Immunofluorescence staining was performed to evaluate the effects of shikonin on actin ring formation. The expression levels of the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway were determined by Western blot analysis. To determine whether shikonin influences the receptor activator of nuclear factor-κB ligand (RANKL)-induced association between receptor activator of NF-κB (RANK) and tumor necrosis factor receptor associated factor 6 (TRAF6), immunofluorescence staining and immunoprecipitation experiments were performed. During our validation model, histomorphometric examination and micro-computed tomography (CT) were conducted to assess the morphology of osteoporosis.. Shikonin prevented bone loss by inhibiting osteoclastogenesis in vitro and improving bone loss in ovariectomized mice in vivo. At the molecular level, Western blot analysis indicated that shikonin inhibited the phosphorylation of inhibitor of NF-κB (IκB), P50, P65, extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and P38. Interaction of TRAF6 and RANK was prevented, and downstream MAPK and NF-κB signaling pathways were downregulated.. Osteoclastic bone resorption was reduced in the presence of shikonin in vitro and in vivo. Shikonin is a promising candidate for treatment of postmenopausal osteoporosis.

Topics: Animals; Biomarkers; Bone Resorption; Cell Differentiation; Cell Survival; Disease Models, Animal; Disease Susceptibility; Female; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Immunohistochemistry; Mice; Mitogen-Activated Protein Kinases; Models, Biological; Naphthoquinones; NF-kappa B; Osteoclasts; Osteogenesis; Osteoporosis, Postmenopausal; Ovariectomy; Protein Binding; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; TNF Receptor-Associated Factor 6; X-Ray Microtomography

Oxidative stress acts as the major causative factor for various age-associated neurodegenerative diseases, triggering cognitive and memory impairments. In the present study, the underlying neuroprotective mechanism governing how shikonin acts against D-galactose (D-gal)-induced memory impairment, neuroinflammation and neuron damage was examined. The results revealed that chronic administration of D-gal [150 mg/kg intraperitoneally (i.p.)] in mice caused cognitive and memory impairments, as determined by Morris water-maze test. Shikonin treatment, however, alleviated D-gal-induced memory impairment and reversed the D-gal-induced neural damage and apoptosis. Furthermore, western blotting and the results of morphological analysis revealed that shikonin treatments markedly reduced D-gal induced neuroinflammation through inhibition of astrocytosis as determined by glial fibrillary acidic protein (GFAP) detection, and downregulating other inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. Moreover, shikonin treatment led to inhibition of the activation of nuclear factor-κB (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs), preventing neurodegeneration. Hence, taken together, the results of the present study suggested that shikonin attenuated D-gal-induced memory impairment, neuroinflammation and neurodegeneration, possibly via the NF-κB/mitogen-activated protein kinase (MAPK) pathway. Our data suggest that shikonin could be a promising, endogenous and compatible antioxidant candidate for age-associated neurodegenerative diseases, including Alzheimer's disease.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Cognitive Dysfunction; Cytokines; Extracellular Signal-Regulated MAP Kinases; Galactose; Humans; Inflammation Mediators; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; Neurodegenerative Diseases; Neurogenic Inflammation; NF-kappa B; Oxidative Stress; Signal Transduction

Nasopharyngeal carcinoma (NPC) is a malignant tumor which is commonly found in East Asia and Africa. The present clinical treatment of NPC is still mainly based on chemotherapeutics and is prone to drug resistance and adverse reactions. Shikonin has been demonstrated to play the antitumor effect in various cancers. However, the specific effects and related regulatory mechanism of Shikonin in NPC have not been clearly declared yet. Cell viability was valued through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell proliferation was detected through colony formation assay and Bromodeoxyuridine (BrdU) assay. Hochest 33258 staining was used to value cell apoptosis. Cell migration and invasion were valued through wound healing and transwell invasion assay, respectively. Glucose uptake, lactate release, ATP level and pyruvate kinase M2 isoform (PKM2) activity were measured using corresponding assay kits. Western blotting was used to examine the expression of proteins related to cell proliferation, cell apoptosis, cell migration and the phosphatidylinositol 3 kinase (PI3K)/AKT signal pathway. We found that Shikonin treatment effectively suppressed cell proliferation and induced obvious cell apoptosis compared with the control. Besides, Shikonin treatment suppressed cell migration and invasion effectively. The detection about glycolysis showed that Shikonin treatment suppressed cell glucose uptake, lactate release and ATP level. The activity of PKM2 was also largely inhibited by Shikonin. Further study revealed that the PI3K/AKT signal pathway was inactivated by Shikonin treatment. In addition, the inducer of the PI3K/AKT signal pathway largely abolished the antitumor effect of Shikonin on cell proliferation, cell apoptosis, cell mobility and aerobic glycolysis in NPC cells. Shikonin inhibits growth and invasion of NPC cells through inactivating the PI3K/AKT signal pathway.

Topics: Apoptosis; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Drugs, Chinese Herbal; Glycolysis; Humans; Naphthoquinones; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Shikonin is a natural naphthoquinone derivative that specifically occurs in boraginaceous plants, and the major active ingredient of the medicinal plant Lithospermum erythrorhizon. Previously, a cytochrome P450 oxygenase (CYP) CYP76B74 catalyzing 3″-hydroxylation of geranylhydroquinone (GHQ) - a key intermediate of shikonin biosynthesis, was identified from cultured cells of Arnebia euchroma. However, the enzymes catalyzing oxidation of the geranyl side-chain of GHQ from L. erythrorhizon remain unknown. In this study, we performed transcriptome analysis of different tissues (red roots and green leaves/stems) from L. erythrorhizon using RNA sequencing technology. Highly expressed CYP genes found in the roots were then heterologously expressed in Saccharomyces cerevisiae and functionally screened with GHQ as the substrate. As the result, two CYPs of CYP76B subfamily catalyzing the oxidation of GHQ were characterized. CYP76B100 catalyzed the hydroxylation of the geranyl side-chain of GHQ at the C-3″ position to form 3″-hydroxyl geranylhydroquinone (GHQ-3″-OH). The enzyme CYP76B101 carried out oxidation reaction of GHQ at the C-3″ position to produce a 3″-carboxylic acid derivative of GHQ (GHQ-3″-COOH) as well as GHQ-3″-OH. This enzyme-catalyzed oxidation reaction with GHQ as the substrate is reported for the first time. This study implicates CYP76B100 and CYP76B101 as having a potential role in shikonin biosynthesis in L. erythrorhizon.

Topics: Cytochrome P-450 Enzyme System; Lithospermum; Naphthoquinones; Terpenes

Shikonin, a natural plant pigment, is known to have anti-obesity activity and to improve insulin sensitivity. This study aimed to examine the effect of shikonin on hepatic steatosis, focusing on the AMP-activated protein kinase (AMPK) and energy expenditure in Hepa 1-6 cells and in high-fat fed mice. Shikonin increased AMPK phosphorylation in a dose- and time-dependent manner, and inhibition of AMPK with compound C inhibited this activation. In an oleic acid-induced steatosis model in hepatocytes, shikonin suppressed oleic acid-induced lipid accumulation, increased AMPK phosphorylation, suppressed the expression of lipogenic genes, and stimulated fatty acid oxidation-related genes. Shikonin administration for four weeks decreased body weight gain and the accumulation of lipid droplets in the liver of high-fat fed mice. Furthermore, shikonin promoted energy expenditure by activating fatty acid oxidation. In addition, shikonin increased the expression of PPARγ coactivator-1α (PGC-1α), carnitine palmitoyltransferase-1 (CPT1) and other mitochondrial function-related genes. These results suggest that shikonin attenuated a high fat diet-induced nonalcoholic fatty liver disease by stimulating fatty acid oxidation and energy expenditure via AMPK activation.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carnitine O-Palmitoyltransferase; Cells, Cultured; Diet, High-Fat; Disease Models, Animal; Dose-Response Relationship, Drug; Energy Metabolism; Fatty Liver; Gene Expression; Lipid Metabolism; Mice; Naphthoquinones; Oxidation-Reduction; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphorylation; Phytotherapy

The objective of this study was to investigate the possible interaction of shikonin and β,β-dimethylacrylshikonin (DSK) with tramadol.. Human liver microsome (HLM) and rat liver microsome (RLM) incubation experiments were carried out to assess the half-maximal inhibitory concentration (IC. The results of this study suggest that shikonin and DSK can inhibit tramadol metabolism both in vitro and in vivo.

Topics: Administration, Oral; Analgesics, Opioid; Animals; Anthraquinones; Dose-Response Relationship, Drug; Drug Interactions; Humans; Inhibitory Concentration 50; Male; Microsomes, Liver; Naphthoquinones; Rats; Rats, Sprague-Dawley; Tramadol

Unruptured ectopic pregnancy (UEP) is a common cause of morbidity and, occasionally, of mortality in women of reproductive age. Pharmacological intervention is a common therapeutic approach for early-stage UEP. Herein, we investigated the cytotoxic effect and novel mechanism of shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, in human trophoblast cells. These data demonstrated that shikonin suppressed proliferation and induced apoptosis in a time-dependent manner in HTR-8/SVneo cells. Shikonin blocked autophagic flux and promoted p62 interaction with caspase 8, resulting in caspase 8 activation. Moreover, shikonin suppressed GLI1 expression, and GLI1 overexpression attenuated shikonin-induced cell apoptosis. Although silencing GLI1 slightly promoted cell apoptosis, p62 overexpression enhanced GLI1 silencing-induced cell apoptosis by activating caspase 8. Furthermore, rapamycin increased shikonin-induced cell apoptosis in HTR-8/SVneo cells, whereas 3-MA attenuated the cytotoxic effect of shikonin. In conclusion, shikonin suppressed trophoblast cell growth by silencing GLI1 and increasing p62 co-mediated activation of caspase 8, which suggested a potential novel therapeutic target for UEP.

Topics: Apoptosis; Autophagy; Caspase 8; Cell Line; Cell Proliferation; Cell Survival; Humans; Naphthoquinones; RNA-Binding Proteins; Trophoblasts; Zinc Finger Protein GLI1

The aim of the present study was to identify the anticancer effects and the mechanisms of action of shikonin and its analogue isobutyrylshikonin in oral squamous carcinoma cells.. The cytotoxic effects of isobutyrylshikonin and shikonin in Ca9-22 and SCC-25 cells were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry analysis of Annexin V/Propidium Iodide (PI) staining, western blot analysis and immunohistochemistry.. Treatment with both isobutyrylshikonin and shikonin induced dose- and time-dependent apoptotic cell death in Ca9-22 cells, although the IC. The present study suggest that isobutyrylshikonin may be a more potent chemotherapeutic agent against oral cancer cells than shikonin. In addition, our data exhibit that both isobutyrylshikonin and shikonin induce caspase-dependent apoptosis via the mitochondrial pathway through accumulation of ROS in oral squamous carcinoma cells.

Topics: Apoptosis; Cell Line, Tumor; Humans; Membrane Potential, Mitochondrial; Mouth Neoplasms; Naphthoquinones; Reactive Oxygen Species; Tumor Cells, Cultured

Postmenopausal osteoporosis is very common in women. Currently, many kinds of new drugs are being developed for this disease. Postmenopausal osteoporosis is closely related to overactivity of osteoclasts in body. Shikonin is purple red naphthoquinone pigment extracted from lithospermum, which has anti-inflammation, antivirus, anticancer and other bioactivities. At the same time, it has been proved that shikonin can promote the proliferation and differentiation of osteoblasts, but its influence on osteoclasts and molecular mechanism are unknown. Our study showed that shikonin could inhibit the activity and formation of RANKL-mediated osteoclasts depending on dose without affecting the activity of bone marrow macrophages (BMM). In addition, we have also found that shikonin can inhibit the expression of specific marker gene of osteoclasts, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAcP) and calcitonin receptor. Shikonin also could promote the proliferation of MC3T3-E1, increasing the expression of mRNA related to osteogenesis, like the expression of bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). Luciferase reporter gene assay and Western blot analysis further indicated that shikonin could inhibit the activity of RANKL-induced NF-κB and NFAT receptors. Moreover, shikonin can also slow down bone loss of ovariectomized (OVX) mice by inhibiting the activity of osteoclasts. This work explains the molecular mechanism of shikonin in RANKL-mediated formation of osteoclasts, and reveals the potential of further developing shikonin into a new drug for prevention and treatment of postmenopausal osteoporosis.

Topics: Animals; Bone Resorption; Cell Differentiation; Mice; Naphthoquinones; NF-kappa B; NFATC Transcription Factors; Osteoclasts; Osteogenesis; Osteoporosis

This study will investigate the effect of shikonin on the proliferation and apoptosis of human ovarian cancer cell SKOV3 (HOCC-SKOV3).. We will retrieve potential studies from inception to the March 1, 2020 in Cochrane Library, MEDLINE, EMBASE, Scopus, Cumulative Index to Nursing and Allied Health Literature, WANGFANG, and China National Knowledge In-frastructure. There are not restrictions related to the language and publication status. This study will include case-controlled studies (CCSs) or randomized controlled studies (RCSs) that examine the effect of shikonin on the proliferation and apoptosis of HOCC-SKOV3. Two researchers will independently identify literatures, extract data, and appraise study quality. Any disagreements will be resolved by discussion with another researcher. RevMan 5.3 software will be placed to perform statistical analysis.. This study will summarize the present evidence to test the effect of shikonin on the proliferation and apoptosis of HOCC-SKOV3.. It will provide evidence to investigate the effect of shikonin on the proliferation and apoptosis of HOCC-SKOV3, and will supply reference for further study.Systematic review registration: INPLASY202040146.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cystadenocarcinoma, Serous; Female; Humans; Meta-Analysis as Topic; Naphthoquinones; Ovarian Neoplasms; Systematic Reviews as Topic

Topics: Antifungal Agents; Aspergillus; Drug Resistance, Fungal; Humans; Naphthoquinones; Proteomics; Reactive Oxygen Species; Tandem Mass Spectrometry

Shikonin induced necroptosis in Jurkat cells were identified flow cytometrically by the up-regulation of RIP3 in live cells and that a proportion of these cells underwent other forms of regulated cell death (RCD) which included parthanatos (< 10%), or cleaved PARP (< 10%) and DNA Damage (> 30%). Live necroptotic cells also possessed functioning mitochondria with hyper-polarized mitochondria membrane potential and generated a fivefold increase in cellular reactive oxygen species (ROS) which was resistant to inhibition by zVAD and necrostatin-1 (Nec-1). After loss of plasma membrane integrity these dead necroptotic cells then showed a higher incidence of parthanatos (> 40%), or cleaved PARP (> 15%) but less DNA Damage (< 15%). Inhibition of shikonin induced apoptosis and necroptosis by zVAD and Nec-1 respectively resulted in live necroptotic cells with an increased incidence of cleaved PARP and reduced levels of DNA Damage respectively. Dead necroptotic cells then showed a reduced incidence of parthanatos and DNA Damage after inhibition by zVAD and Nec-1 respectively. A high proportion of these dead necroptotic cells (30%) which lacked plasma membrane integrity also displayed functioning hyper-polarized mitochondria with high levels of cellular ROS and thus had the capacity to influence the outcome of RCD processes rather than just been the end product of cell death, the necrotic cell. Flow cytometry can thus measure multiple forms of RCD and the level of cellular ROS and MMP which highlights the inter-connection between cell death processes and that a single cell may simultaneously display multiple forms of RCD.

Topics: Antineoplastic Agents; Apoptosis; Cell Proliferation; DNA Damage; Flow Cytometry; Gene Expression Regulation, Neoplastic; Histones; Humans; Imidazoles; Indoles; Jurkat Cells; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Necroptosis; Nuclear Pore Complex Proteins; Oligopeptides; Parthanatos; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; RNA-Binding Proteins; Signal Transduction

Zicao is the dried root of Lithospermum erythrorhizon Sieb, et Zucc, Arnebia euchroma (Royle) Johnst, or Arnebia guttata Bunge and commonly used to treat viral infection, inflammation, arthritis and cancer in China.Shikonin (SKN) is a major active chemical component isolated from zicao. Previous research showed that SKN has anti-inflammatory, immunomodulatory and analgesic effects, and inhibits the development of arthritis and the condition of collagen arthritis (CIA) mice; nevertheless, its role in the angiogenesis of rheumatoid arthritis (RA) has not been elucidated.. The purpose of this study was to investigate the antiangiogenic activity of SKN in CIA rats and various angiogenesis models.. The anti-arthritic effect of SKN on CIA rats was tested by arthritis score, arthritis incidence, radiological observation and histopathology evaluation of inflamed joints. Vessel density evaluated with CD31 immunohistochemistry/immunofluorescence in joint synovial membrane tissues of CIA rats, chick chorioallantoic membrane assay, rat aortic ring assay, and the migration, invasion, adhesion and tube formation of human umbilical vein endothelial (HUVEC) cells induced by tumor necrosis factor (TNF)-α were used to measured the antiangiogenenic activity of SKN. Moreover, the effect of SKN on the expression of angiogenic mediators, such as vascular endothelial growth factor (VEGF), VEGFR2, TNF-α, interleukin (IL)-1β, platelet derived growth factor (PDGF) and transforming growth factor (TGF)-β in sera and joint synovia of rats, and in TNF-α-induced MH7A/HUVEC cells were measured by immunohistochemistry, enzyme linked immunosorbent assay, Western blot and/or real-time polymerase chain reaction (PCR). Through the analysis of protein and mRNA levels of phosphoinositide 3-kinase (PI3K), Akt and PTEN, and the autophosphorylation of ERK1/2, JNK and p38 in joint synovia of rats and in TNF-α-induced HUVEC cells, the molecular mechanism of its inhibition was elucidated by using Western blot and/or real-time PCR.. These findings indicate for the first time that SKN has the anti-angiogenic effect in RA in vivo, ex vivo and in vitro by interrupting the PI3K/AKT and MAPKs signaling pathways.

Topics: Angiogenesis Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Arthritis, Rheumatoid; Chick Embryo; Chorioallantoic Membrane; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Male; MAP Kinase Signaling System; Naphthoquinones; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley

Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1β production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Gene Knockdown Techniques; Humans; Macrophages; Mice; Naphthoquinones; Pyruvate Kinase; Rats; RAW 264.7 Cells; RNA, Small Interfering; Severity of Illness Index; Signal Transduction; STAT1 Transcription Factor; Synovial Membrane

Shikonin, one of the main active ingredients of Chinese herbal medicine Lithospermum erythrorhizon, has been widely used to treat various disease including virus infection and inflammation in clinical. Its anti-tumor activity has been recorded in "Chinese herbal medicine". Recently, some studies about its anti-glioma effects have been reported. However, little is known about the molecular pharmacological activity of Shikonin in glioma.. This study aimed to systematically uncover and validate the pharmacological mechanism of Shikonin against glioma.. Network pharmacology approach, survival analysis, and Pearson co-expression analysis were performed to uncover and test the pharmacological mechanisms of Shikonin in glioma. Apoptosis assay, Caspase-3 activity assay and immunoblot analysis were practiced to validate the mechanisms.. Network pharmacology results suggested, anti-glioma effect of Shikonin by interfering endoplasmic reticulum (ER) stress-mediated tumor apoptosis targeting Caspase-3, and Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP) triggering cancer cell apoptosis. Survival analysis suggested the association of CASP3 with glioma (P < 0.05). Pearson correlation analysis indicated possible interaction of CASP3 with PERK through positive feedback regulation. Shikonin or in combination with 14G2a induced cell apoptosis in oligodendroglioma Hs683 cells in a dose-dependent manner with at a maximum apoptosis rate of 33%-37.5%, and 73%-77% respectively. Immunoblot analysis showed that Shikonin increased Caspase-3 activity to about 4.29 times, and increased 9 times when it combined with 14G2a. Shikonin increased also the expression levels of the proteins PERK and CHOP by about 4.4 and 5.6 folds, respectively, when it combined with 14G2a.. This study highlights the pharmacological mechanisms of Shikonin in the induction of tumor apoptosis in glioma cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Membrane Permeability; Endoplasmic Reticulum Stress; Gene Regulatory Networks; Glioma; Humans; Mitochondrial Membranes; Naphthoquinones

Psoriasis is a chronic inflammatory skin disease characterized by well‑defined scaly papules and plaques. Interleukin (IL)‑17 is involved in its pathogenesis and promotes the proliferation of epidermal keratinocytes through signal transducer and activator of transcription 3 (STAT3) activation. Shikonin, a natural naphthoquinone isolated from Lithospermum erythrorhizon, possesses anti‑inflammatory and immunosuppressive properties and can suppress IL‑17‑induced vascular endothelial growth factor expression by inhibiting the JAK/STAT3 pathway. In the present study, MTS, iCELLigence and RT‑qPCR were used to determine the optimal concentration and duration of IL‑17 or shikonin acting on HaCaT cells. The changes in the expression levels of genes associated with the IL‑6/STAT3 pathway in differentially treated cells were analyzed via RT2Profiler™ PCR Array. Small interfering RNA was used to silence the expression levels of the target gene CCAAT/enhancer‑binding protein δ (CEBPD). Western blotting and immunohistochemistry were used to evaluate the effect of shikonin on imiquimod‑induced psoriasis in mice and the expression levels of CEBPD. Shikonin reversed IL‑17‑mediated downregulation of the tumor suppressor CEBPD in HaCaT cells. Moreover, low levels of CEBPD in the imiquimod‑induced mouse model of psoriasis were restored by shikonin treatment, which ameliorated excessive keratinocyte proliferation. Taken together, these findings suggest that CEBPD plays a key role in the pathogenesis of psoriasis and can be targeted by shikonin as a potential therapeutic strategy.

Topics: Animals; CCAAT-Enhancer-Binding Protein-delta; Cell Proliferation; Disease Models, Animal; Down-Regulation; HaCaT Cells; Humans; Imiquimod; Interleukin-17; Interleukin-6; Mice; Naphthoquinones; Psoriasis; Signal Transduction; STAT3 Transcription Factor

Increasing evidence indicates that the inflammatory tumor microenvironment can lead to cancer cell metastasis. Shikonin, which is extracted from the Chinese herb Zicao (the dried root of Lithospermum erythrorhizon), possesses various pharmacological effects, but its effect on tumor metastasis in the inflammatory microenvironment remains unknown. In the present study, we aimed to investigate the potential effect of shikonin on tumor metastasis in an inflammatory microenvironment as well as the underlying molecular mechanisms. It was found that, in the inflammatory microenvironment simulated by THP‑1 cell conditioned medium (THP‑1‑CM) in vitro, shikonin significantly inhibited the epithelial‑mesenchymal transition (EMT), migration and invasion of human lung adenocarcinoma cell lines A549 and H1299. In addition, we found that interleukin‑6 (IL‑6), which is expressed in THP‑1‑CM, promoted the EMT of lung adenocarcinoma cells, and shikonin markedly inhibited IL‑6‑induced EMT and cell motility. Moreover, shikonin inhibited IL‑6‑induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), prevented phosphorylated STAT3 (p‑STAT3) translocation into the nucleus, and suppressed p‑STAT3 transactivation activity. Additionally, it was found that shikonin inhibited lung metastasis, EMT and expression of p‑STAT3 of A549 cells in vivo. Furthermore, IL‑6 levels in human lung adenocarcinoma tissues were significantly associated with tumor‑node‑metastasis stage and lymph node metastasis, and its expression was correlated with tumor‑associated macrophage (TAM) infiltration. Together, these results suggest that shikonin suppresses the migration and invasion of human lung adenocarcinoma cells in an inflammatory microenvironment involving the IL‑6/STAT3 signaling pathway.

Topics: A549 Cells; Adenocarcinoma of Lung; Cell Movement; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Female; Humans; Interleukin-6; Lung; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Naphthoquinones; Neoplasm Invasiveness; Signal Transduction; STAT3 Transcription Factor; THP-1 Cells; Tumor Microenvironment; Tumor-Associated Macrophages; Xenograft Model Antitumor Assays

One of the most significant adverse postburn responses is abnormal scar formation, such as keloids. Despite its prolificacy, the underlying pathophysiology of keloid development is unknown. We recently demonstrated that NLRP3 inflammasome, the master regulator of inflammatory and metabolic responses (e.g., aerobic glycolysis), is essential for physiological wound healing. Therefore, burn patients who develop keloids may exhibit altered immunometabolic responses at the site of injury, which interferes with normal healing and portends keloid development. Here, we confirmed keloid NLRP3 activation (cleaved caspase-1 [P < 0.05], IL-1β [P < 0.05], IL-18 [P < 0.01]) and upregulation in Glut1 (P < 0.001) and glycolytic enzymes. Burn skin similarly displayed enhanced glycolysis and Glut1 expression (P < 0.01). However, Glut1 was significantly higher in keloid compared with nonkeloid burn patients (>2 SD above mean). Targeting aberrant glucose metabolism with shikonin, a pyruvate kinase M2 inhibitor, dampened NLRP3-mediated inflammation (cleaved caspase-1 [P < 0.05], IL-1β [P < 0.01]) and improved healing in vivo. In summary, burn skin exhibited evidence of Warburg-like metabolism, similar to keloids. Targeting this altered metabolism could change the trajectory toward normal scarring, indicating the clinical possibility of shikonin for abnormal scar prevention.

Topics: Adult; Animals; Anti-Inflammatory Agents, Non-Steroidal; Burns; Case-Control Studies; Female; Glucose Transporter Type 1; Glycolysis; Humans; Inflammasomes; Inflammation; Inflammation Mediators; Keloid; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Naphthoquinones; NLR Family, Pyrin Domain-Containing 3 Protein; Pyruvate Kinase; Skin; Wound Healing

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.

Topics: Alcohol Dehydrogenase; Aldehydes; Benzoquinones; Lithospermum; Metabolic Networks and Pathways; Naphthoquinones; Terpenes

Colorectal cancer (CRC) is a common malignancy occurring in the digestive system. Despite progress in surgery and therapy options, CRC is still a considerable cause of cancer mortality worldwide. In this study, a colon cancer patient-derived xenograft model was established to evaluate the antitumor activity of Shikonin. The protective effect underlying Shikonin was determined through assessing serum levels of liver enzymes (ALT, AST) and kidney functions (BuN, Scr) in PDX mice. Proteomics and metabolomics profiles were integrated to provide a systematic perspective in dynamic changes of proteins and global endogenous metabolites as well as their perturbed pathways. A total of 456 differently expressed proteins (DEPs), 32 differently expressed metabolites (DEMs) in tumor tissue, and 20 DEMs in mice serum were identified. The perturbation of arginine biosynthesis, purine metabolism, and biosynthesis of amino acids may mainly account for therapeutic mechanism of Shikonin. Furthermore, the expression of mRNAs participating in arginine biosynthesis (CPS1, OTC, Arg1) and do novo purine synthesis (GART, PAICS, ATIC) were validated through RT-qPCR. Our study provides new insights into the drug therapeutic strategies and a better understanding of antitumor mechanisms that might be valuable for further studies on Shikonin in the clinical treatment of colorectal cancer.

Topics: Alanine Transaminase; Animals; Antineoplastic Agents; Aspartate Aminotransferases; Blood Urea Nitrogen; Cell Cycle Checkpoints; Cell Proliferation; Colonic Neoplasms; Creatinine; Female; Humans; Metabolomics; Mice; Mice, Nude; Naphthoquinones; Neoplasm Proteins; Proteomics; Xenograft Model Antitumor Assays

Recent research suggests that melatonin (Mel), an endogenous hormone and natural supplement, possesses anti-proliferative effects and can sensitise cells to anti-cancer therapies. Although shikonin (SHK) also possesses potential anti-cancer properties, the poor solubility and severe systemic toxicity of this compound hinders its clinical usage. In this study, we combined Mel and SHK, a potentially promising chemotherapeutic drug combination, with the aim of reducing the toxicity of SHK and enhancing the overall anti-cancer effects. We demonstrate for the first time that Mel potentiates the cytotoxic effects of SHK on cancer cells by inducing oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway. Particularly, Mel-SHK treatment induced oxidative stress, increased mitochondrial calcium accumulation and reduced the mitochondrial membrane potential in various cancer cells, leading to apoptosis. This drug combination also promoted endoplasmic reticulum (ER) stress, leading to AKT dephosphorylation. In HeLa cells, Mel-SHK treatment reduced SIRT3/SOD2 expression and SOD2 activity, while SIRT3 overexpression dramatically reduced Mel-SHK-induced oxidative stress, ER stress, mitochondrial dysfunction and apoptosis. Hence, we propose the combination of Mel and SHK as a novel candidate chemotherapeutic regimen that targets the SIRT3/SOD2-AKT pathway in cancer.

Topics: Apoptosis; Cell Death; HeLa Cells; Humans; Melatonin; Naphthoquinones; Neoplasms; Oxidative Stress; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Sirtuin 3; Superoxide Dismutase

Pyruvate kinase M2 (PKM2) is an enzyme that is predominantly overexpressed in various types of cancer. The role of PKM2 in liver fluke-associated cholangiocarcinoma (CCA) remains unclear. This study aimed to investigate the antitumor activity of shikonin, a PKM2 inhibitor, in CCA cells.. Immunohistochemistry and immunoblotting were used to determine PKM2 expression in CCA tissues and cells. Antiproliferative effects of shikonin were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation and trypan blue exclusion assays. The anti-metastatic activity of shikonin was determined using the Boyden chamber assay. Mechanisms by which shikonin inhibited CCA progression were determined.. PKM2 was overexpressed in CCA compared to normal bile duct epithelial cells. Shikonin significantly inhibited growth, and migration of CCA cells while inducing their death. A mechanistic study revealed that antitumor effects of shikonin in CCA cells depended on increased production of reactive oxygen species.. Shikonin may be a novel therapeutic agent for patients with CCA.

Topics: Antineoplastic Agents; Apoptosis; Bile Duct Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Humans; Membrane Proteins; Naphthoquinones; Reactive Oxygen Species; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Multiple sclerosis is a common autoimmune inflammatory disease of the central nervous system. There are several underlying mechanisms for the pathogenesis of the disease, including inflammation, oligodendrocyte apoptosis and oxidative stress.. The mechanism of action of shikonin was investigated in the C57BL/6 experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.. The results revealed that EAE induction significantly increased the extent of demyelination in the corpus callosum tissues of the animals, while treatment of the mice with shikonin significantly decreased the extent of demyelination. Real-time polymerase chain reaction-based analysis of the brain samples from the EAE mice revealed significant enhancement in the expression levels of tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and Bax genes as well as a reduction in the expression levels of transforming growth factor-ß (TGF-β) and Bcl2. But, shikonin treatment significantly reduced the expression levels of TNF-α, IFN-γ and Bax. On the other hand, the expression levels of TGF-β and Bcl2 as well as the activity of glutathione peroxidase-1 (GPX-1) enzyme were significantly increased following the shikonin treatment.. This study emphasized the immune-modulatory and antioxidative effects of shikonin, which may have an important healing effect on the severity of EAE.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Corpus Callosum; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Immunologic Factors; Interferon-gamma; Mice, Inbred C57BL; Naphthoquinones; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno-associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well-known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood-derived CD34 + HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype-dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector-mediated transgene expression. Taken together, these studies expand our understanding of rAAV6-mediated transduction in hematopoietic cells and are informative for improving rAAV6-based treatment of blood diseases.

Topics: Cells, Cultured; Dependovirus; Genetic Vectors; Hematopoietic Stem Cells; Humans; Leupeptins; Medicine, Chinese Traditional; Naphthoquinones; Parvovirinae; Proteasome Endopeptidase Complex; Reactive Oxygen Species; Transduction, Genetic

Cancer immunotherapy has been a favorable strategy for facilitating antitumor immunity. However, immune tolerance and an ultimate immunosuppressive tumor microenvironment (ITM) are primary obstacles. To achieve the goals of remodeling the ITM and promoting cancer immunotherapy, a versatile nanoparticle codelivering shikonin (SK) and PD-L1 knockdown siRNA (SK/siR-NPs) was reported. SK/siR-NPs are demonstrated to tellingly induce the immunogenic cell death (ICD) of tumor cells, leading to increased dendritic cell maturation. Moreover, SK/siR-NPs can cause an efficacious inhibition of PD-L1, leading to enhanced cytotoxic T lymphocyte response to tumor cells. Most importantly, SK/siR-NPs can restrain lactate production via the downregulation of pyruvate kinase-M2 (PKM2) and eventually repolarize tumor associated macrophages (TAMs) from the M2-subtype to M1-subtype states. Meanwhile, SK/siR-NPs suppress regulatory T lymphocytes to fight with the ITM. Overall, the developed co-delivery system presents a significant potential for cancer immunotherapy through simultaneously inducing ICD, repolarizing M2-TAMs, and relieving PD-L1 pathway-regulated immune tolerance.

Topics: Animals; Cell Death; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Female; Immunosuppressive Agents; Immunotherapy; Mice; Mice, Inbred BALB C; Multifunctional Nanoparticles; Naphthoquinones; Particle Size; RNA, Small Interfering; Surface Properties; Tumor Cells, Cultured; Tumor Microenvironment

To observe the effect of shikonin on the proliferation, migration, adhesion and invasion of rheumatoid arthritis(RA) fibroblast like synoviocytes induced by tumor necrosis factor-α(TNF-α), and to explore its mechanism of action from aspects of protein kinase B(Akt) and mitogen activated protein kinase(MAPK) signaling pathways. TNF-α(20 ng·mL~(-1)) was used in this experiment to induce human RA fibroblast like synovial cell line(MH7 A). After addition of different concentrations of shikonin(0.025, 0.05, 0.1 pmol·L~(-1)), the proliferation, migration, adhesion and invasion ability of MH7 A cells were detected by MTT test, scratch test, adhesion test, Transwell invasion test, respectively. Protein expression of Akt and MAPK signaling pathway molecules in MH7 A cells was detected by Western blot. The results showed that as compared with the control group, TNF-α could significantly induce the proliferation, migration, adhesion and invasion of MH7 A cells, and increase the phosphorylation level of Akt, JNK, p38 and extracellular regulatory protein kinase(ERK). As compared with the TNF-α group, shikonin had no significant effect on TNF-α-induced proliferation of MH7 A cells after 24 h treatment, and it could reduce the TNF-α-induced proliferation of MH7 A cells in a concentration dependent manner after 48 h treatment. Shikonin also significantly reduced the TNF-α-induced migration, adhesion, invasion and phosphorylation levels of Akt, JNK, p38, ERK in MH7 A cells within 24 h. These results suggested that shikonin could reduce the proliferation, migration, adhesion and invasion ability of MH7 A cells induced by TNF-α, and its mechanism may be related to the down-regulation of Akt and MAPK signaling pathway activation.

Topics: Arthritis, Rheumatoid; Cell Movement; Cell Proliferation; Cells, Cultured; Fibroblasts; Humans; Naphthoquinones; Synovial Membrane; Synoviocytes; Tumor Necrosis Factor-alpha

In this study, we aim to investigate whether shikonin prevents against NAFLD. After feeding high-fat diet (HFD) for 10 weeks, Sprague-Dawley rats were received different doses of shikonin (5mg/kg/day, 10mg/kg/day and 20mg/kg/day) by gavage for the last 12 weeks of a total of 22 weeks of a HFD. Our results showed that total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol, aspartate aminotransferase and alanine aminotransferase were significantly increased, while high-density lipoprotein cholesterol was decrease, accompanied by hepatic injury and lipid accumulation in HFD-fed rats. Shikonin treatment attenuated the above biochemical and histopathological changes. Similarly, HFD-induced the increase of hepatic TC and TG levels were also ameliorated by shikonin treatment. Furthermore, shikonin observably mitigated HFD-induced the liver fibrosis and the increase of plasminogen activator inhibitor type 1, connective tissue growth factor, collagen III and IV expression. Additionally, shikonin markedly inhibited HFD-induced the decrease of proliferator-activated receptor γ (PPARγ) and matrix metalloproteinases-9 (MMP-9) expression and the increase of tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in liver tissue. This study demonstrates that shikonin ameliorates hepatic lipid dysregulation and fibrosis through PPARγ and MMP-9/TIMP-1 axis, suggesting that shikonin may be a potential therapeutic agent for the treatment of NAFLD.

Topics: Animals; Body Weight; Boraginaceae; Diet, High-Fat; Gene Expression Regulation; Lipid Metabolism; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Naphthoquinones; Non-alcoholic Fatty Liver Disease; Plants, Medicinal; PPAR gamma; Rats, Sprague-Dawley; Tissue Inhibitor of Metalloproteinase-1

Development of antifungal agents with novel mechanism and low toxicity are essential due to the prevalence of the infectious diseases caused by

Topics: Acetylation; Antifungal Agents; Biological Factors; Candida albicans; Chromatography, Liquid; Gas Chromatography-Mass Spectrometry; Histones; Lysine; Metabolomics; Naphthoquinones; Protein Processing, Post-Translational; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

In this study, we developed an AS1411 aptamer/hyaluronic acid-bifunctionalized microemulsion co-loading shikonin and docetaxel (AS1411/SKN&DTX-M). Such microemulsion was capable of penetrating the blood-brain barrier (BBB), targeting CD44/nucleolin-overexpressed glioma, and inhibiting the orthotopic glioma growth. AS1411/SKN&DTX-M showed a spherical morphology with a diameter around 30 nm and rapidly released drugs in the presence of hyaluronidase and mild acid. In the U87 cellular studies, AS1411/SKN&DTX-M elevated the cytotoxicity, enhanced the cellular uptake, and induced the cell apoptosis. In the artificial blood-brain barrier model, the transepithelial electrical resistance was decreased after the treatment with AS1411/SKN&DTX-M and thereby of increasing the apparent permeability coefficient. Furthermore, AS1411/SKN&DTX-M showed a strong inhibition against the formation of cancer stem cell-enriched U87 cell spheroids, in which the expression of CD133 was downregulated significantly. In the biodistribution studies, AS1411/SKN&DTX-M could selectively accumulate in the brains of orthotopic luciferase-transfected U87 glioma tumor-bearing nude mice. Importantly, AS1411/SKN&DTX-M exhibited the overwhelming inhibition of glioma growth of orthotopic luciferase-transfected U87 glioma models and reached the longest survival period among all the treatments. In summary, the codelivery of shikonin and docetaxel using bifunctionalization with hyaluronic acid and AS1411 aptamer offers a promising strategy for dual drug-based combinational antiglioma treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aptamers, Nucleotide; Cell Line; Cell Line, Tumor; Docetaxel; Drug Delivery Systems; Emulsions; Glioma; Humans; Hyaluronic Acid; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Naphthoquinones; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; RNA-Binding Proteins; Tissue Distribution

To investigate the role of inflammatory response, oxidative damage and changes of ATP-sensitive potassium channels (sK. Time course studies on inflammatory response by real-time PCR, oxidative process and function of isolated basilar artery after SAH in New Zealand White rabbits were performed. Basilar artery smooth muscle cells (BASMCs) in each group were obtained and whole-cell patch-clamp technique was applied to record cell membrane capacitance and K. Inflammatory cytokines levels were highest at 24h compare to 72h after SAH whereas the oxidative damage and cell death marker were at highest peak at 72h. Oxidative damage peak coincided with significant alterations in cell membrane capacitance, K. Currents of ATP-sensitive potassium channels in basilar smooth muscle cells decreased after SAH by putative oxidative modification from immediate inflammatory response and can be protected by shikonin pretreatment.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Basilar Artery; Cytokines; Female; Inflammation Mediators; KATP Channels; Male; Muscle, Smooth, Vascular; Naphthoquinones; Oxidative Stress; Patch-Clamp Techniques; Rabbits; Subarachnoid Hemorrhage

Endophytic fungi are microorganisms located in the inter- or intracellular compartments of plant tissues but with no harmful effects. They are considered a potential source of biological compounds. The present study was conducted to investigate the molecular identification of endophytic fungi isolated from the roots of Lithospermum officinale and their potential production of shikonin. Phylogenetic analysis was performed based on the Internal Transcribed Spacer (ITS) region and the isolates were classified into five genera as follows: Alternaria, Chaetosphaeronema, Fusarium, Mucor, and Trichoderma. The study on the methanol extracts of endophytic fungi indicated that total polyphenol content had a positive relationship with antioxidant activities and the highest antioxidant activity belonged to the methanol extracts of Fusarium tricinctum and Alternaria altenata. Then, to investigate the ability of the fungal isolates to produce shikonin, a naphthoquinone compound with high biological activity, the extracts were subjected to HPLC. The results obtained from HPLC-mass spectrometry showed that shikonin could be produced only by F. tricinctum. Thus, F. tricinctum isolated from the roots of L. officinale can be presented as a new source of shikonin.

Topics: Antioxidants; Biphenyl Compounds; Drug Evaluation, Preclinical; Endophytes; Lithospermum; Naphthoquinones; Phylogeny; Phytochemicals; Picrates; Plant Roots

Chromatinolysis refers to enzymatic degradation of nuclear DNA and is regarded as one of the crucial events leading to cell death. Mixed-lineage kinase domain-like protein (MLKL) has been identified as a key executor of necroptosis, but it remains unclear whether MLKL contributes to necroptosis via regulation of chromatinolysis. In this study, we find that shikonin induces MLKL activation and chromatinolysis in glioma cells in vitro and in vivo, which are accompanied with nuclear translocation of AIF and γ-H2AX formation. In vitro studies reveal that inhibition of MLKL with its specific inhibitor NSA or knockdown of MLKL with siRNA abrogates shikonin-induced glioma cell necroptosis, as well as chromatinolysis. Mechanistically, activated MLKL targets mitochondria and triggers excessive generation of mitochondrial superoxide, which promotes AIF translocation into nucleus via causing mitochondrial depolarization and aggravates γ-H2AX formation via improving intracellular accumulation of ROS. Inhibition of nuclear level of AIF by knockdown of AIF with siRNA or mitigation of γ-H2AX formation by suppressing ROS with antioxidant NAC effectively prevents shikonin-induced chromatinolysis. Then, we found that RIP3 accounts for shikonin-induced activation of MLKL, and activated MLKL reversely up-regulates the protein level of CYLD and promotes the activation of RIP1 and RIP3. Taken together, our data suggest that MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis, and shikonin induces a positive feedback between MLKL and its upstream signals RIP1 and RIP3.

Topics: Animals; Apoptosis Inducing Factor; Cell Line, Tumor; DNA Fragmentation; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Mitochondria; Naphthoquinones; Necroptosis; Protein Kinases; Rats; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cell Movement; Cell Proliferation; Cells, Cultured; Fibroblasts; Fibronectins; Gene Expression; Gingiva; Humans; Lactate Dehydrogenases; MAP Kinase Signaling System; Naphthoquinones; Vascular Endothelial Growth Factor A; Wound Healing

Shikonin, a natural naphthoquinone, has attracted much attention due to its various biological activities. Two shikonin glucosides, shikonin-1',8-di-O-β-D-glucopyranoside (1) and shikonin-1'-O-β-D-glucopyranoside (2), were biosynthesized through in vitro enzymatic glycosylation and their structures were elucidated using spectroscopic techniques. The water-solubility and stability of compounds 1 and 2 were significantly higher than those of the parent compound. Furthermore, compound 2 showed moderate cytotoxicity against six cancer cell lines, with IC

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Glucosides; Glycosylation; Glycosyltransferases; Humans; Hydrogen-Ion Concentration; Molecular Structure; Naphthoquinones; Solubility; Structure-Activity Relationship; Temperature

Shikonin is a naphthoquinone compound extracted from the root of Lithospermum with various pharmacological activities. Sympathetic neural remodeling greatly contributes to chronic heart failure. Growing evidence has identified a critical role of microRNAs (miRNAs) in a variety of cardiac biological processes. This study aimed to verify whether shikonin could attenuate sympathetic neural remodeling and explore the possible regulatory role of miRNAs in this process.. Shikonin was administered to mice after transverse aortic constriction (TAC). Immunohistochemistry and western blotting were used to assess the expression of TAC-induced sympathetic remodeling-related proteins.. TAC-induced expression of the sympathetic remodeling-related proteins, tyrosine hydroxylase (TH), growth associated protein 43 (GAP43), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and nerve growth factor (NGF), was significantly decreased in cardiac tissues. MiR-124 expression significantly increased after heart failure and decreased after shikonin treatment. An adeno-associated virus 9 (AAV9) vector was packaged and used to transfect myocardial tissues of aortic-constricted mice with miR-124, resulting in increased heart miR-124 levels and inhibition of the effects of shikonin on sympathetic neural remodeling. Immunohistochemical staining showed that the density of TH-, GAP43-, and ChAT-positive nerves was significantly increased in aortic-constricted mice after transfection with AAV9-miR-124.. Our data demonstrate that shikonin administration prevents sympathetic neural remodeling in mice with TAC-induced heart failure. The effects of shikonin on heart failure may be partly due to miR-124-mediated attenuation of sympathetic remodeling. Our results reveal a novel mechanism underlying the therapeutic effect of shikonin in heart failure.

Topics: Animals; Cardiotonic Agents; Chronic Disease; Constriction, Pathologic; GAP-43 Protein; Gene Expression Regulation; Heart Failure; Male; Mice, Inbred C57BL; MicroRNAs; Myocardium; Naphthoquinones; Sympathetic Nervous System; Vesicular Acetylcholine Transport Proteins

Topics: A549 Cells; Acrylates; Animals; Antineoplastic Agents; Apoptosis; Benzoates; Drug Design; ErbB Receptors; Humans; Mice, Nude; Molecular Docking Simulation; Naphthoquinones; Neoplasms; Tubulin; Tubulin Modulators

Colon cancer is the second most common deadliest malignancy in the world and better understanding of its underlying mechanisms is needed to improve clinical management. Natural plant extracts are gaining attention in the development of new therapeutic strategies against various cancer types. Shikonin is a naturally extracted naphthoquinone pigment with effects against cancer, including colon cancer.. In this study, we conducted a series of in vitro experiments to show the effects of Shikonin on colon cancer cell apoptosis. A colon cancer cell line with overexpression of peroxiredoxin V (PrxV) was constructed and the relationship of PrxV expression with Shikonin-induced cell apoptosis was investigated.. Shikonin induced colon cancer cell apoptosis via regulation of mammalian target of rapamycin signaling. Shikonin-induced cell apoptosis was abrogated by overexpression of PrxV.. According to the results obtained in this study, targeting PrxV may provide new insight for the successful management of colon cancer by inducing cell apoptosis.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Naphthoquinones; Peroxiredoxins; Tumor Cells, Cultured

Liver fibrosis is a worldwide clinical challenge during the progression of chronic liver disease to liver cirrhosis. Shikonin is extracted from the root of Lithospermum erythrorhizon with antioxidant, anti-inflammatory, anticancer, and wound-healing properties. The study aims to investigate the protective effect of shikonin on liver fibrosis and its underlying mechanism.. Shikonin significantly inhibited activation of hepatic stellate cells and extracellular matrix formation by downregulating the transforming growth factor-β1 expression and maintaining the normal balance between metalloproteinase-2 and tissue inhibitor of metalloproteinase-1. Shikonin also decreased hepatic stellate cell energy production by inhibiting autophagy.. The results confirmed that shikonin attenuated liver fibrosis by downregulating the transforming growth factor-β1/Smads pathway and inhibiting autophagy.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartate Aminotransferases; Autophagy; Disease Models, Animal; Down-Regulation; Extracellular Matrix; Hepatic Stellate Cells; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Naphthoquinones; Signal Transduction; Smad Proteins, Receptor-Regulated; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.

Topics: Biosynthetic Pathways; Cluster Analysis; Gene Expression Regulation, Plant; Lithospermum; Naphthoquinones; Plant Proteins; Proteome; Proteomics; Reproducibility of Results; Sequence Analysis, RNA

Fatty acid synthase (FASN) catalyzing the terminal steps in the de novo biogenesis of fatty acids is correlated with low survival and high disease recurrence in patients with bladder cancer. Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels and provides a growth advantage to tumors. However, it is unclear whether the change of PKM2 has an effect on FASN and what is the mechanisms underlying. Here we describe a novel function of PKM2 in control of lipid metabolism by mediating transcriptional activation of FASN, showing the reduced expression of sterol regulatory element binding protein 1c (SREBP-1c). We first discovered that PKM2 physically interacts with the SREBP-1c using biochemical approaches, and downregulation of PKM2 reduced the expression of SREBP-1c by inactivating the AKT/mTOR signaling pathway, which in turn directly suppressed the transcription of major lipogenic genes FASN to reduce tumor growths. Furthermore, either PKM2 inhibitor-Shikonin or FASN inhibitor-TVB-3166 alone induced a strong antiproliferative and anticolony forming effect in bladder cancer cell line. The combination of both inhibitors exhibits a super synergistic effect on blocking the bladder cancer cells growth. It provides a new target and scientific basis for the treatment of bladder cancer.

Topics: Azetidines; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Fatty Acid Synthase, Type I; Gene Expression Regulation, Neoplastic; Humans; Lipogenesis; Membrane Proteins; Naphthoquinones; Nitriles; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Thyroid Hormone-Binding Proteins; Thyroid Hormones; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Shikonin, a naphthoquinone derivative isolated from the root of

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Carrier Proteins; Cell Proliferation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Liver Neoplasms; Membrane Proteins; Mitochondria; Molecular Structure; Naphthoquinones; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Signal Transduction; Structure-Activity Relationship; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Topics: Biosynthetic Pathways; Lithospermum; Naphthoquinones; Proteomics

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP1B1; Drug Screening Assays, Antitumor; HCT116 Cells; Hep G2 Cells; Humans; K562 Cells; Mice; Naphthoquinones; Prodrugs; Xenograft Model Antitumor Assays

Shikonin and its derivatives are the most abundant naphthoquinone pigments formed in species of the medicinally and economically valuable Boraginaceae. A key step in the shikonin biosynthetic pathway, namely the C-3'' hydroxylation of the prenylated phenolic intermediate geranylhydroquinone, is expected to be catalyzed by a cytochrome P450. To identify cytochrome P450 candidates with transcription profiles similar to those of genes known to be involved in shikonin biosynthesis, we carried out coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines and examined the spatial expression of candidate genes in different organs of

Topics: Boraginaceae; Cytochrome P-450 Enzyme System; Endoplasmic Reticulum; Hydroxylation; Naphthoquinones; Oryza; Phylogeny; Plant Proteins; Plant Roots; RNA Interference; Saccharomyces cerevisiae; Terpenes

Shikonin, a natural naphthoquinone, is abundant in Chinese herb medicine Zicao (purple gromwell) and has a wide range of biological activities, especially for cancer. Shikonin and its analogues have been reported to induce cell-cycle arrest, but target information is still unclear. We hypothesized that shikonin, with a structure similar to that of quinone-type compounds, which are inhibitors of cell division cycle 25 (Cdc25) phosphatases, will have similar effects on Cdc25s. To test this hypothesis, the effects of shikonin on Cdc25s and cell-cycle progression were determined in this paper.. The in vitro effects of shikonin and its analogues on Cdc25s were detected by fluorometric assay kit. The binding mode between shikonin and Cdc25B was modelled by molecular docking. The dephosphorylating level of cyclin-dependent kinase 1 (CDK1), a natural substrate of Cdc25B, was tested by Western blotting. The effect of shikonin on cell cycle progression was investigated by flow cytometry analysis. We also tested the anti-proliferation activity of shikonin on cancer cell lines by MTT assay. Moreover, in vivo anti-proliferation activity was tested in a mouse xenograft tumour model.. Shikonin and its analogues inhibited recombinant human Cdc25 A, B, and C phosphatase with IC. In this study, we provide evidence for how shikonin induces cell cycle arrest and functions as a Cdc25s inhibitor. It shows an anti-proliferation effect both in vitro and in vivo by mediating Cdc25s.

Topics: Animals; cdc25 Phosphatases; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Drugs, Chinese Herbal; Humans; Mice; Molecular Targeted Therapy; Naphthoquinones; Reactive Oxygen Species; Recombinant Proteins; Xenograft Model Antitumor Assays

Breast cancer is the most common malignancy in women. Apoptosis is important for tumor suppression and may delay cancer progression. It was found that shikonin induced apoptosis in 4T1 murine mammary cancer cells and MDA‑MB‑231 human breast cancer cells in vitro. Total p38 and c‑Jun N‑terminal kinase (JNK) levels were maintained in 4T1 cells, and p38 phosphorylation, but not JNK phosphorylation, was significantly increased. Caspase‑3/7 activity was detected, which suggested that the p38 pathway, but not the JNK signaling pathway, induced apoptosis in 4T1 cells. The anti‑tumor effects of shikonin on orthotopic mouse models were also examined. On day 7 after inoculation of 4T1 cells into mice, tumor volumes in the shikonin‑treated and the control groups began to differ. On day 13, tumors were weighed, and shikonin was revealed to suppress tumor growth in the orthotopic 4T1 model in vivo. In conclusion, shikonin is a potential anti‑tumor drug for breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Drug Screening Assays, Antitumor; Female; Humans; Mammary Neoplasms, Experimental; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphorylation

Abnormal activation of neddylation modification and dysregulated energy metabolism are frequently seen in many types of cancer cells. Whether and how neddylation modification affects cellular metabolism remains largely unknown. Here, we showed that MLN4924, a small-molecule inhibitor of neddylation modification, induces mitochondrial fission-to-fusion conversion in breast cancer cells via inhibiting ubiquitylation and degradation of fusion-promoting protein mitofusin 1 (MFN1) by SCFβ-TrCP E3 ligase and blocking the mitochondrial translocation of fusion-inhibiting protein DRP1. Importantly, MLN4924-induced mitochondrial fusion is independent of cell cycle progression, but confers cellular survival. Mass-spectrometry-based metabolic profiling and mitochondrial functional assays reveal that MLN4924 inhibits the TCA cycle but promotes mitochondrial OXPHOS. MLN4924 also increases glycolysis by activating PKM2 via promoting its tetramerization. Biologically, MLN4924 coupled with the OXPHOS inhibitor metformin, or the glycolysis inhibitor shikonin, significantly inhibits cancer cell growth both in vitro and in vivo. Together, our study links neddylation modification and energy metabolism, and provides sound strategies for effective combined cancer therapies.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclopentanes; Energy Metabolism; Female; GTP Phosphohydrolases; HEK293 Cells; Humans; Metformin; Mice; Mitochondria; Mitochondrial Dynamics; Mitochondrial Membrane Transport Proteins; Naphthoquinones; Neoplasms; Oxidative Phosphorylation; Proteolysis; Pyrimidines; Ubiquitin-Activating Enzymes; Ubiquitination; Xenograft Model Antitumor Assays

Acetaminophen (APAP) overdose causes acute liver injury and leads to fatal liver damage. However, the therapies are quite limited. Shikonin is a natural product with antioxidant and anti-inflammatory activities. In the present study, the hepatoprotective effects and the underlying mechanisms of shikonin in APAP-induced hepatotoxicity in vivo and in vitro were investigated. APAP-induced acute liver injury and shikonin pretreatment models were established in vivo and in vitro, as evidenced by serum hepatic enzymes, histological changes, oxidative stress indicators and proinflammatory cytokines. The results revealed that shikonin pretreatment prevented the elevation of serum alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) levels and markedly reduced APAP-induced histological alterations in liver tissues. Additionally, shikonin restored superoxide dismutase (SOD) expression and glutathione (GSH) content in line with the blockade of oxidative stress. The changes in gene expression involved in oxidative stress including methionine sulfoxide reductase (such as MsrA and MsrB1), heme oxygenase-1 (HO-1), SOD2 and cytochrome P450 2E1 (CYP2E1), were markedly reversed after shikonin therapy. Furthermore, shikonin markedly attenuated the APAP-induced production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) and suppressed the expression of genes related to inflammation. In AML-12 cells, shikonin pretreatment decreased APAP-induced cytotoxicity as measured by CCK-8 assay and LDH release. The changes in gene expression involved in oxidative stress and the inflammatory response were consistent with those in mouse livers. This study indicated that shikonin attenuated APAP-induced acute liver injury via inhibiting oxidative stress and inflammatory responses in vivo and in vitro. These findings offer new insights into the potential therapy for APAP hepatotoxicity.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Anti-Inflammatory Agents, Non-Steroidal; Chemical and Drug Induced Liver Injury; Inflammation; Male; Mice; Mice, Inbred BALB C; Naphthoquinones; Oxidative Stress

Shikonin, a botanical drug extracted from Lithospermum erythrorhizon, exhibits anti-cancer effects in various cancer cell lines. However, the mechanisms underlying these effects have not been completely elucidated yet. Here, we showed that Shikonin induces apoptosis and autophagy in A375 cells and inhibits their proliferation. Shikonin caused G2/M phase arrest through upregulation of p21 and downregulation of cyclin B1. Shikonin significantly triggered ER stress-mediated apoptosis by upregulating the expression of p-eIF2α, CHOP, and cleaved caspase-3. It also induced protective autophagy by activating the p38 pathway, followed by an increase in the levels of p-p38, LC3B-II, and Beclin 1. Upon suppression of autophagy by 3-methyladenine, Shikonin-induced apoptosis was enhanced in A375 cells. Moreover, after pretreatment with N-acetyl-cysteine, Shikonin increased the production of reactive oxygen species that are involved in regulating ER stress-mediated apoptosis and p38-activated autophagy, as evidenced by the reversion of cell viability and apoptosis and a decrease in p-eIF2α, CHOP, p-p38, LC3B-II, and Beclin 1 levels. Thus, we demonstrated that Shikonin induced apoptosis and autophagy in A375 cells via the activation of ROS-mediated ER stress and p38 pathways, indicating that Shikonin can serve as a potential agent for human melanoma therapy.

Topics: Apoptosis; Autophagy; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Stress; G2 Phase Cell Cycle Checkpoints; Humans; MAP Kinase Signaling System; Melanoma; Molecular Structure; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species

Shikonin, a traditional Chinese medicine, has been identified as being capable of inducing apoptosis in various tumors, including glioma, and is thus considered to be a promising therapeutic agent for tumor therapy. However, little is known about the molecular mechanism of shikonin in glioma. The present study investigated the influence of shikonin on the proliferation and apoptosis of glioma cells U251 and U87MG and explored the potential molecular mechanisms. It was identified that shikonin was able to induce apoptosis in human glioma cells in a time‑ and dose‑dependent manner, and a decreased expression level of cluster of differentiation (CD)147 was observed in shikonin‑treated U251 and U87MG cells. Knockdown of CD147 inhibited U251 and U87MG cell growth, whereas CD147 overexpression enhanced cell growth and decreased shikonin‑induced apoptosis. Additionally, an increased expression level of CD147 suppressed the elevated production of reactive oxygen species and mitochondrial membrane potential levels induced by shikonin. The data indicated that shikonin‑induced apoptosis in glioma cells was associated with the downregulation of CD147 and the upregulation of oxidative stress. CD147 may be an optional target of shikonin‑induced cell apoptosis in glioma cells.

Topics: Apoptosis; Basigin; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Glioma; Humans; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Naphthoquinones; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering

Remodeling tumor immune microenvironment (TIME) is an important strategy to lift the immunosuppression and achieve immune normalization. In this work, a mannosylated lactoferrin nanoparticulate system (Man-LF NPs) is developed for dual-targeting biomimetic codelivery of shikonin and JQ1 via the mannose receptor and LRP-1 that are overexpressed in both cancer cells and tumor-associated macrophages. The Man-LF NPs can serve as multitarget therapy for inducing immune cell death in the cancer cells, repressing glucose metabolism and repolarizing tumor-associated macrophages, and consequently, lead to remodeling the TIME (e.g., promotion of dendritic cell maturation and CD8

Topics: Azepines; Biomimetics; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Dendritic Cells; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Lactoferrin; Lectins, C-Type; Low Density Lipoprotein Receptor-Related Protein-1; Macrophages; Mannose; Mannose Receptor; Mannose-Binding Lectins; Nanoparticles; Naphthoquinones; Neoplasms; Receptors, Cell Surface; Triazoles; Tumor Microenvironment

Renal interstitial fibrosis is a common pathological feature of chronic kidney disease that may involve changes of metabolism in kidney cells. In the present study, we first showed that blockade of glycolysis with either dichloroacetate (DCA) or shikonin to target different glycolytic enzymes reduced renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO). Both inhibitors evidently suppressed the induction of fibronectin and collagen type I in obstructed kidneys, with DCA also showing inhibitory effects on collagen type IV and α-smooth muscle actin (α-SMA). Histological examination also confirmed less collagen deposition in DCA-treated kidneys. Both DCA and shikonin significantly inhibited renal tubular apoptosis but not interstitial apoptosis in UUO. Macrophage infiltration after UUO injury was also suppressed. Shikonin, but not DCA, caused obvious animal weight loss during UUO. To determine whether shikonin and DCA worked on tubular cells and/or fibroblasts, we tested their effects on cultured renal proximal tubular BUMPT cells and renal NRK-49F fibroblasts during hypoxia or transforming growth factor-β

Topics: Animals; Apoptosis; Cell Line; Dichloroacetic Acid; Disease Models, Animal; Enzyme Inhibitors; Epithelial Cells; Extracellular Matrix; Fibroblasts; Fibrosis; Glycolysis; Kidney Diseases; Kidney Tubules; Macrophages; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; Signal Transduction; Ureteral Obstruction

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Count; Cell Differentiation; Forkhead Transcription Factors; Immune Tolerance; Mice; Naphthoquinones; Proto-Oncogene Proteins c-akt; Psoriasis; RNA, Messenger; Skin; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

Triple-negative breast cancer (TNBC) is characterized by elevated metastasis, low survival, and poor response to therapy. Although many specific and effective agents for treating TNBC have been investigated, promising therapeutic options remain elusive. Here, we screened the inhibitory activities of three main components of Lithospermum erythrorhizon Sieb. et Zucc (shikonin, acetylshikonin, and β,β-dimethylacrylshikonin) on TNBC cells. The results revealed that shikonin potently decreased the viabilities of TNBC MDA-MB-231 and 4T1 cells but showed less cytotoxicity to normal mammary epithelial MCF-12A cells. Additionally, shikonin reversed the epithelial-to-mesenchymal transition (EMT) in MDA-MB-231 and 4T1 cells. Shikonin depressed cell migration and invasion, upregulated E-cadherin levels, downregulated N-cadherin, vimentin, and Snail levels, and reorganized the cytoskeletal proteins F-actin and vimentin. Shikonin reversed EMT by inhibiting activation of β-catenin signaling through attenuating β-catenin expression, nuclear accumulation, binding to T-cell factor consensus oligos, and transcription of its targeted EMT-related genes. Moreover, shikonin upregulated glycogen synthase kinase 3β (GSK-3β) levels, leading to enhanced phosphorylation and decreased levels of β-catenin. Furthermore, shikonin administration significantly inhibited lung metastasis of MDA-MB-231 cells in NOD/SCID mice accompanied by low systemic toxicity. Histological analysis confirmed that shikonin elevated levels of E-cadherin, phosphorylated β-catenin, and GSK-3β, and decreased levels of vimentin and β-catenin in pulmonary metastatic foci. These results indicated that shikonin potently inhibits TNBC metastasis by targeting the EMT via GSK-3β-regulated suppression of β-catenin signaling, which highlights the importance of shikonin as a potential candidate for novel anticancer therapeutics against TNBC.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; beta Catenin; Cell Line, Tumor; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Female; Glycogen Synthase Kinase 3 beta; Mice; Mice, Inbred NOD; Mice, SCID; Naphthoquinones; Signal Transduction; Triple Negative Breast Neoplasms

Shikonin is an active compound of the oriental medicinal plant, Leptospermum erythrorhizon, which has been previously shown to inhibit psoriasis-like inflammation. However, the underlying mechanism is unclear. In the present study, the mechanisms of keratinocyte proliferation and apoptosis in psoriasis in response to shikonin were explored both in vitro and in vivo. Our results showed that shikonin significantly inhibits cell proliferation and induces apoptosis in both HaCaT and LV-STAT3 HaCaT cells by targeting CEBPD, while a decrease in cell survival, proliferation and viability were found through flow-cytometry and MTS assay. Furthermore, gavage with shikonin markedly alleviated psoriasis-like manifestations in IMQ-induced BALB/c mice clinically (PASI Score) and histopathologically. Immunohistochemistry revealed that shikonin potently suppresses the JAK/STAT3 signaling pathway in local skin lesions and increases CEBPD expression. These results imply that shikonin inhibits keratinocyte proliferation and induces apoptosis, which results in psoriasis treatment through the JAK/STAT3 dependent pathway. In addition, the activation of JAK/STAT3 downregulates CEBPD in HaCaT cells and IMQ-induced BALB/c mice. However, shikonin can reverse these effects, suggesting that CEBPD may be a potential therapeutic target for psoriasis.

Topics: Animals; Apoptosis; CCAAT-Enhancer-Binding Protein-delta; Cell Line; Cell Proliferation; Humans; Imiquimod; Janus Kinases; Keratinocytes; Male; Mice, Inbred BALB C; Naphthoquinones; Psoriasis; STAT3 Transcription Factor; Up-Regulation

Shikonin, a natural red colorant, is widely used for food garnishment and cosmetic ingredient in the world. Shikonin also possesses a variety of pharmacological actions, including anti-inflammation and anti-cancer activities. However, little is known about its effects on the UDP-glucuronosyltransferases (UGT) activity. Therefore, the aim of this study was to evaluate the effect of shikonin on the UGT1A1, UGT1A3, UGT1A6, UGT1A9 and UGT2B7 activities via the human and rat liver microsomal assay and cocktail approach. The results showed shikonin inhibited human and rat liver microsomal UGT activity only in a dose-dependent manner. The further enzyme kinetic studies demonstrated that shikonin was not only a competitive inhibitor of human UGT1A1, UGT1A9, and UGT2B7, but also presented competitive inhibition on rat UGT1A1 and AZTG reactions. In conclusion, shikonin as a reversible inhibitor of UGT enzyme has a high-risk potential to cause the possible toxicity, especially drug-drug or food-drug interactions.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glucuronosyltransferase; Humans; Male; Microsomes, Liver; Molecular Structure; Naphthoquinones; Rats; Rats, Sprague-Dawley; Species Specificity

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Carrier Proteins; Dimethyl Sulfoxide; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Glycolysis; Lymphopoiesis; Membrane Proteins; Mice, Inbred C57BL; Naphthoquinones; Th1 Cells; Th17 Cells; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Bladder cancer is a "Warburg-like" tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.. HTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values.. The three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects.. Our studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Carrier Proteins; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Drugs, Chinese Herbal; Female; Glucose; Glycolysis; Humans; Male; Membrane Proteins; Middle Aged; Naphthoquinones; Protein Structure, Quaternary; Pyruvate Kinase; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Urinary Bladder Neoplasms

Echium plantagineum, a native of Europe and Africa, is a noxious invasive weed in Australia forming monocultural stands in pastures and rangelands. It produces a complex mixture of bioactive secondary metabolites, including toxic pyrrolizidine alkaloids (PAs), that protect the plant from insect and livestock herbivory and naphthoquinones (NQs), which suppress competition from weeds, insects and pathogens, and also influence invasion success. However, the extent to which allelochemical production is impacted by environmental factors, thereby influencing plant defense against pests, remains unclear.. Following plant stress induced by drought, herbivory and high temperature, extracts of E. plantagineum shoots and roots were subjected to metabolic profiling by UPLC-MS-DAD- QToF mass spectrometry. Abundance of NQs, especially deoxyshikonin, shikonin and dimethylacrylshikonin, rapidly increased in roots exposed to elevated temperatures. Water withholding initially increased NQ abundance, but prolonged drought resulted in reduced total PAs and NQs. Intraspecific competition elevated the production of NQs, whereas simulated herbivory had no initial effect on NQs. Following herbivory, the abundance of the PA 3'-O-acetylechimidine-N-oxide in seedling shoots was increased.. Differential accumulation of defense metabolites by E. plantagineum following exposure to various stressors suggested stress-dependent biosynthetic regulation, particularly with respect to NQ production, which was rapidly induced following drought, intraspecific competition and high temperature treatment, thereby positively impacting resistance or defense against herbivores, weeds and pathogens. We propose that trade-offs between above- and below-ground metabolism in E. plantagineum may facilitate allelochemical production in response to stress, rendering plants with an enhanced ability to defend against other neighboring plants, insects and microbes, with allelochemical production further facilitated by catabolic recycling following lengthier exposure to stress. © 2019 Society of Chemical Industry.

Topics: Echium; Naphthoquinones; Plant Weeds; Pyrrolizidine Alkaloids; Stress, Physiological

Pathological scarring is an intractable problem for both patients and clinicians. A major obstacle for the development of scar remediation therapies is the paucity of suitable in vivo and in vitro models. The "Scar-in-a-jar" model was previously established by our colleagues based on the principle of "Macromolecular crowding". This has been demonstrated to be an extracellular matrix-rich in vitro model offering a novel tool for studies related to the extracellular matrix. In the study reported herein, we have optimised this approach to model human dermal fibroblasts derived from hypertrophic tissues. This optimised in vitro model has been found to hold similar properties, such as increased collagen I, interleukins and transforming growth factor beta-1 expression, compared to that observed in hypertrophic scar tissue in vivo. In addition, Shikonin has been previously demonstrated to hold potential as a novel hypertrophic scar treatment due to its apoptosis-inducing property on hypertrophic scar fibroblasts. Other Shikonin analogues have also been reported to hold apoptosis-inducing properties in various cancer cell lines, however, the effects of these analogues on hypertrophic scar-related cells are unknown. We therefore evaluated the effects of Shikonin and its analogues on hypertrophic scar-derived human fibroblasts using the optimised "Macromolecular crowding" model. Our data indicates that Shikonin and Naphthazarin are the most effective molecules compared to related naphthoquinones. The data generated from the study offers a novel in vitro collagen-rich model of hypertrophic scar tissue. It also provides further evidences supporting the use of Shikonin and Naphthazarin as potential treatments for hypertrophic scars.

Topics: Animals; Apoptosis; Cell Line; Cicatrix; Cicatrix, Hypertrophic; Collagen; Extracellular Matrix; Fibroblasts; Humans; Models, Biological; Naphthoquinones; Skin

Pseudoshikonin I (PSI), a novel biomaterial isolated from Lithospermi radix, has been recognized as an herbal medicine for the treatment of infectious and inflammatory diseases. Bone remodeling maintains a balance through bone resorption (osteoclastogenesis) and bone formation (osteoblastogenesis). Bone formation is generally attributed to osteoblasts. However, the effects of PSI on the bone are not well known. In this study, we found that the ethanol extracts of PSI induced osteoblast differentiation by increasing the expression of bone morphogenic protein 4 (BMP 4). PSI positively regulates the transcriptional expression and osteogenic activity of osteoblast-specific transcription factors such as Runx2 and Osterix. To identify the signaling pathways that mediate PSI-induced osteoblastogenesis, we examined the effects of serine-threonine kinase inhibitors that are known regulators of Osterix and Runx2. PSI-induced upregulation of Osterix and Runx2 was suppressed by treatment with AKT and PKA inhibitors. These results suggest that PSI enhances osteoblast differentiation by stimulating Osterix and Runx2 via the AKT and PKA signaling pathways. Thus, the activation of Runx2 and Osterix is modulated by PSI, thereby demonstrating its potential as a treatment target for bone disease.

Topics: Animals; Bone Morphogenetic Protein 4; Bone Remodeling; Cell Differentiation; Cell Line; Core Binding Factor Alpha 1 Subunit; Ethanol; Gene Expression Regulation; HEK293 Cells; Humans; Lithospermum; Mice; Naphthoquinones; Osteoblasts; Plant Extracts; Sp7 Transcription Factor; Transcription, Genetic

Shikonin (SHK) is a highly liposoluble naphthoquinone pigment has recently been investigated as a potential antiglioma agent. However, shikonin shows several limitations like poor aqueous solubility, short half-life and non-selective biodistribution. Herein, we have developed a nanoparticles (NPs) prepared from PEG-PLGA using an emulsion solvent evaporation method. Nanoparticle surfaces were modified by coating with lactoferrin (Lf) to improve the crossing of the blood brain barrier and targeting of glioma cells via receptor-mediated path-ways. X-ray powder diffraction and differential scanning calorimetry analysis revealed the amorphous nature of SHK encapsulated within the NPs. Moreover, the drug-loaded NPs exhibit narrow size distribution and high encapsulation efficiency. The in vitro release experiments of the NPs exhibited sustained release for more than 72h. When compared to free SHK and SHK/NPs, in vivo study demonstrated higher brain concentration of SHK, indicating a significant effect of Lf coated NPs on brain targeting. Accordingly, these findings provide evidence for the potential of Lf-modified NPs as a targeted delivery system for brain glioblastomas treatment.

Topics: Animals; Brain; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Glioma; Humans; Lactoferrin; Nanoparticles; Naphthoquinones; Polyesters; Polyethylene Glycols; Rats; Tissue Distribution

Recognition of a new therapeutic agent may activate an alternative programmed cell death for the treatment of breast cancer.. Here, it has been tried to evaluate the effects of Shikonin, a naphthoquinone derivative of Lithospermum erythrorhizon, on the induction of necroptosis and apoptosis mediated by RIPK1-RIPK3 in the ER+ breast cancer cell line, MCF-7.. In the current study, cell death modalities, cell cycle patterns, RIPK1 and RIPK3 expressions, caspase-3 and caspase-8 activities, reactive oxygen species and mitochondrial membrane potential have been evaluated in the Shikonin-treated MCF-7 cells.. Necroptosis and apoptosis have been occurred by Shikonin, with a significant increase in RIPK1 and RIPK3 expressions, although necroptosis was the major rout in MCF-7 cells. Shikonin significantly increased the percentage of the cells in sub-G1 and also those in the later stages of cell cycle, which represents an increase in necroptosis and apoptosis. Under caspase inhibition by Z-VAD-FMK, Shikonin has stimulated necroptosis, which could be arrested by Nec-1. An increase in ROS levels and a decrease in the mitochondrial membrane potential have also been observed.. On the basis of present findings, Shikonin has been suggested as a good candidate for the induction of cell death in ER+ breast cancer, although further investigations, experimental and clinical, are required.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Death; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Molecular Structure; Naphthoquinones; Reactive Oxygen Species; Receptors, Estrogen; Structure-Activity Relationship; Tumor Cells, Cultured

In current study, a series of shikonin derivatives were synthesized and its anticancer activity was evaluated. As a result, PMMB232 showed the best antiproliferation activity with an IC

Topics: Antineoplastic Agents; Apoptosis; Carboxylic Acids; Coumarins; Drug Evaluation, Preclinical; Female; HeLa Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Molecular Docking Simulation; Naphthoquinones; Uterine Cervical Neoplasms

Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown. In this study, we isolated a cDNA encoding LeMRP, an ATP-binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real-time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes. Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real-time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up-regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down-regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots. Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.

Topics: ATP-Binding Cassette Transporters; Cloning, Molecular; Gene Expression Regulation, Plant; Lithospermum; Membrane Transport Proteins; Naphthoquinones; Phylogeny; Plant Proteins; Plants, Genetically Modified; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA

To examine specific molecular mechanisms involved in modulating hepatic lipogenesis and mitochondria biogenesis signals by Lithospermum erythrorhizon (gromwell) root extract.. Stable cell lines with luciferase reporter constructs were generated to examine sterol regulatory element binding protein 1c (SREBP1c) and peroxisome proliferator-activated receptor gamma, coactivator 1 (PGC1) α promoter activity and estrogen-related receptor (ERR) α response element activity. Gene expression of SREBP1c, stearoyl coenzyme A desaturase 1, and PGC1α was measured by using reverse transcription polymerase chain reaction. Lipogenesis was measured in human hepatoma cells with Nile red staining and flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) α was determined by using ELISA and Western blot.. Gromwell root extract and its naphthoquinones dose-dependently repressed high glucose and liver X receptor α induction of SREBP1c promoter activity and gene expression. Hepatic lipogenesis was repressed, and PGC1α promoter and gene expression and ERRα response element activity were increased by gromwell root extract. Gromwell root extract, shikonin, and α-methyl-n-butyrylshikonin increased AMPKα phosphorylation, and inhibition of AMPK blunted the repression in SREBP1c promoter activity by gromwell root extract and its naphthoquinones.. Data suggest that gromwell root extract and its naphthoquinones repress lipogenesis by increasing the phosphorylated state of AMPKα and stimulating mitochondrial biogenesis signals.

Topics: AMP-Activated Protein Kinases; Animals; CHO Cells; Cricetulus; Hep G2 Cells; Humans; Lithospermum; Naphthoquinones; Sterol Regulatory Element Binding Protein 1; Transfection

Programmed necrosis, necroptosis, is considered to be a highly immunogenic activity, often mediated via the release of damage-associated molecular patterns (DAMPs). Interestingly, enhanced macroautophagic/autophagic activity is often found to be accompanied by necroptosis. However, the possible role of autophagy in the immunogenicity of necroptotic death remains largely obscure. In this study, we investigated the possible mechanistic correlation between phytochemical shikonin-induced autophagy and the shikonin-induced necroptosis for tumor immunogenicity. We show that shikonin can instigate RIPK1 (receptor [TNFRSF]-interacting serine-threonine kinase 1)- and RIPK3 (receptor-interacting serine-threonine kinase 3)-dependent necroptosis that is accompanied by enhanced autophagy. Shikonin-induced autophagy can directly contribute to DAMP upregulation. Counterintuitively, among the released and ectoDAMPs, only the latter were shown to be able to activate the cocultured dendritic cells (DCs). Interruption of autophagic flux via chloroquine further upregulated ectoDAMP activity and resultant DC activation. For potential clinical application, DC vaccine preparations treated with tumor cells that were already pretreated with chloroquine and shikonin further enhanced the antimetastatic activity of 4T1 tumors and reduced the effective dosage of doxorubicin. The enhanced immunogenicity and vaccine efficacy obtained via shikonin and chloroquine cotreatment of tumor cells may thus constitute a compelling strategy for developing cancer vaccines via the use of a combinational drug treatment.

Topics: Alarmins; Animals; Apoptosis; Autophagy; Cell Communication; Cell Line, Tumor; Chloroquine; Dendritic Cells; Female; Immunization; Immunologic Surveillance; Mice, Inbred BALB C; Models, Biological; Naphthoquinones; Necrosis; Neoplasm Metastasis; Up-Regulation

As a continuation of our research on developing potent and potentially safe antineoplastic agents, a set of forty five sulfur-containing shikonin oxime derivatives were synthesized and evaluated for their in vitro cytotoxic activity against human colon cancer (HCT-15), gastric carcinoma (MGC-803), liver (Bel7402), breast (MCF-7) cancer cells and human skin fibroblast (HSF) cells. All the synthesized compounds exhibited potent cytotoxic activity selectively towards HCT-15 cells and did not display apparent toxicity to the normal HSF cells, some of which were more or comparatively effective to the parent compound against HCT-15, MGC-803 and Bel7402 cells. The most active agent 9m displayed high potency against human cancer cells with IC

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Naphthoquinones; Nitric Oxide; Oximes; Structure-Activity Relationship; Sulfur; Tumor Cells, Cultured

Many cancer cells undergo metabolic reprogramming known as the Warburg effect, which is characterized by a greater dependence on glycolysis for ATP generation, even under normoxic conditions. Glyoxalase I (GLO I) is a rate-limiting enzyme involved in the detoxification of cytotoxic methylglyoxal formed in glycolysis and which is known to be highly expressed in many cancer cells. Thus, specific inhibitors of GLO I are expected to be effective anticancer drugs. We previously discovered a novel GLO I inhibitor named TLSC702. Although the strong inhibitory activity of TLSC702 was observed in the in vitro enzyme assay, higher concentrations were required to induce apoptosis at the cellular level. One of the proposed reasons for this difference is that cancer cells alter the energy metabolism leading them to become more dependent on mitochondrial respiration than glycolysis (Metabolic shift) to avoid apoptosis induction. Thus, we assumed that combination of TLSC702 with shikonin-a specific inhibitor of pyruvate kinase M2 (PKM2) that acts as a driver of TCA cycle by supplying pyruvate and which is known to be specifically expressed in cancer cells-would have anticancer effects. We herein show the anticancer effects of combination treatment with TLSC702 and shikonin, and a possible anticancer mechanism.

Topics: Apoptosis; Butyrates; Carrier Proteins; Cell Line, Tumor; Citric Acid Cycle; Drug Screening Assays, Antitumor; Humans; Lactoylglutathione Lyase; Membrane Proteins; Naphthoquinones; Neoplasm Proteins; Neoplasms; Pyruvate Kinase; Pyruvic Acid; Thiazoles; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Acute lung injury (ALI) is a challenging clinical syndrome, which manifests as an acute inflammatory response. Myeloid differentiation protein 2 (MD2) has an important role in mediating LPS-induced inflammation. Currently, there are no effective molecular-based therapies for ALI or viable biomarkers for predicting the severity of disease. Recent preclinical studies have shown that shikonin, a natural naphthoquinone, prevents LPS-induced inflammation. However, little is known about the underlying mechanisms.. The binding affinity of shikonin to MD2 was analysed using computer docking, surface plasmon resonance analysis and elisa. In vitro, the anti-inflammatory effect and mechanism of shikonin were investigated through elisa, real-time quantitative reverse transcription PCR, Western blotting and immunoprecipitation assay. In vivo, lung injury was induced by intratracheal administration of LPS and assessed by changes in the histopathological and inflammatory markers. The underlying mechanisms were investigated by immunoprecipitation in lung tissue.. Shikonin directly bound to MD2 and interfered with the activation of toll-like receptor 4 (TLR4) induced by LPS. In cultured macrophages, shikonin inhibited TLR4 signalling and pro-inflammatory cytokine production. These effects were produced through suppression of key signalling proteins including the NF-κB and the MAPK pathway. We also showed that shikonin inhibits MD2-TLR4 complex formation and reduces LPS-induced inflammatory responses in a mouse model of ALI.. Our studies have uncovered the mechanism underlying the biological activity of shikonin in ALI and suggest that the targeting of MD2 may prove to be beneficial as a treatment option for this condition.

Topics: Acute Lung Injury; Animals; Cytokines; Humans; Inflammation; Lipopolysaccharides; Lymphocyte Antigen 96; MAP Kinase Signaling System; Mice; Molecular Docking Simulation; Naphthoquinones; Toll-Like Receptors

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Drug Design; Glycolysis; HeLa Cells; Humans; Mitosis; Naphthoquinones; Neoplasms; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Tubulin; Tubulin Modulators

Hyperglycemia-induced oxidative stress is thought to play a critical role in the pathogenesis of diabetic nephropathy (DN). Treating high-glucose (HG)-induced proximal tubule injury has become a patential therapeutic option to attenuate the onset and progression of DN. The present study aimed to investigate the renoprotective effect of shikonin, the chief active compound extracted from the roots of the traditional Chinese herb Lithospermum erythrorhizon, on HG-induced cytotoxicity in NRK-52E cells. Treating cells with HG significantly reduce cell viability while also significantly increasing content of reactive oxygen species (ROS). Treating the cells with shikonin improved these changes induced by HG. Shikonin strongly stabilized mitochondrial membrane potential in HG-induced NRK-52E cells. In addition, treatment with shikonin upregulated antioxidant system in response to ROS by increasing levels of SOD and CAT. Furthermore, shikonin also strongly decreased the levels of activated caspase-3, Bax and p-GSK-3β while increased the p-AKT level. These findings provide that the renoprotective effects of shikonin against HG-induced cytotoxicity in NRK-52E cells may be mediated in inhibiting oxidative stress through activating of the AKT signalling pathway.

Topics: Animals; Antioxidants; Apoptosis; Caspase 3; Cell Line; Cell Survival; Diabetic Nephropathies; Epithelial Cells; Glucose; Glycogen Synthase Kinase 3 beta; Kidney Tubules, Proximal; Membrane Potential, Mitochondrial; Naphthoquinones; Oxidative Stress; Protective Agents; Rats; Reactive Oxygen Species; Signal Transduction

Topics: Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Biomarkers; Membrane Potential, Mitochondrial; Molecular Structure; Naphthoquinones; Oxidative Stress; PC12 Cells; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species

Shikonin (SHK) has been proven to have a good anti-tumor effect. However, poor water solubility and low bioavailability limit its wide application in clinical practice. In this study, to overcome these drawbacks, RGD-modified shikonin-loaded liposomes (RGD-SSLs-SHK) were successfully prepared. It exhibited excellent physicochemical characteristics including particle size, zeta potential, encapsulation efficiency, and delayed release time. Meanwhile, the targeting activity of the RGD-modified liposomes was demonstrated by flow cytometry and confocal microscopy in the α

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Adhesion; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Liposomes; MCF-7 Cells; Naphthoquinones; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Transcription Factor RelA

BACKGROUND Shikonin is a component of Chinese herbal medicine. The aim of this study was to investigate the effects of shikonin on cell migration of papillary thyroid cancer cells of the TPC-1 cell line in vitro and expression levels of the phosphate and tensin homolog deleted on chromosome 10 (PTEN) and DNA methyltransferase 1 (DNMT1) genes. MATERIAL AND METHODS The Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the proliferation of TPC-1 papillary thyroid cancer cells, and the normal thyroid cells, HTori-3, in vitro. A transwell motility assay was used to analyze the migration of TPC-1 cells. Western blot was performed to determine the expression levels of PTEN and DNMT1 genes. A methylation-specific polymerase chain reaction (PCR) (MSP) assay was used to evaluate the methylation of PTEN. RESULTS Following treatment with shikonin, the cell survival rate of TPC-1 cells decreased in a dose-dependent manner; the inhibitory effects on HTori-3 cells were less marked. Shikonin inhibited TPC-1 cell migration and invasion in a dose-dependent manner. The methylation of PTEN was suppressed by shikonin, which also reduced the expression of DNMT1 in a dose-dependent manner, and increased the expression of PTEN. Overexpression of DNMT1 promoted the migration of TPC-1 cells and the methylation of PTEN. Levels of protein expression of PTEN in TPC-1 cells treated with shikonin decreased, and were increased by DNMT1 knockdown. CONCLUSIONS Shikonin suppressed the expression of DNMT1, reduced PTEN gene methylation, and increased PTEN protein expression, leading to the inhibition of TPC-1 cell migration.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; DNA (Cytosine-5-)-Methyltransferase 1; Dose-Response Relationship, Drug; Down-Regulation; Gene Amplification; Gene Knockdown Techniques; Humans; Methylation; Naphthoquinones; Neoplasm Invasiveness; PTEN Phosphohydrolase; RNA, Small Interfering; Thyroid Neoplasms

Reliance on aerobic glycolysis is one of the hallmarks of cancer. Although pyruvate kinase M2 (PKM2) is a key mediator of glycolysis in cancer cells, lack of selective agents that target PKM2 remains a challenge in exploiting metabolic pathways for cancer therapy. We report that unlike its structural analog shikonin, a known inhibitor of PKM2, lapachol failed to induce non-apoptotic cell death ferroxitosis in hypoxia. However, melanoma cells treated with lapachol showed a dose-dependent inhibition of glycolysis and a corresponding increase in oxygen consumption. Accordingly, in silico studies revealed a high affinity-binding pocket for lapachol on PKM2 structure. Lapachol inhibited PKM2 activity of purified enzyme as well as in melanoma cell extracts. Blockade of glycolysis by lapachol in melanoma cells led to decreased ATP levels and inhibition of cell proliferation. Furthermore, perturbation of glycolysis in melanoma cells with lapachol sensitized cells to mitochondrial protonophore and promoted apoptosis. These results present lapachol as an inhibitor of PKM2 to interrogate metabolic plasticity in tumor cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Glycolysis; Humans; Melanoma; Mitochondria; Models, Molecular; Naphthoquinones; Oxygen Consumption; Pyruvate Kinase

Shikonin, a naphthoquinone pigment abundant in the root of the Chinese herb Lithospermum erythrorhizon, has been widely used to treat inflammatory diseases for thousands of years. Whether shikonin changes drug metabolism remains unclear.. We investigated whether shikonin modulates the expression of hepatic drug-metabolizing enzymes and transporters as well as the possible mechanisms of this action.. Primary hepatocytes isolated from Sprague-Dawley rats were treated with 0-2 μM shikonin and the protein and mRNA levels of drug-metabolizing enzymes and transporters as well as the activation of aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) were determined.. Shikonin dose-dependently increased the protein and RNA expression of phase I enzymes, i.e., cytochrome P450 (CYP) 1A1/2, CYP3A2, CYP2D1, and CYP2C6; phase II enzymes, i.e., glutathione S-transferase (GST), NADP(H) quinone oxidoreductase 1 (NQO1), and UDP glucuronosyltransferase 1A1; and phase III drug transporters, i.e., P-glycoprotein, multidrug resistance-associated protein 2/3, organic anion transporting polypeptide (OATP) 1B1, and OATP2B1. Immunoblot analysis and EMSA revealed that shikonin increased AhR and Nrf2 nuclear contents and DNA binding activity. AhR and Nrf2 knockdown by siRNA attenuated the ability of shikonin to induce drug-metabolizing enzyme expression. In addition, shikonin increased p38, JNK, and ERK1/2 phosphorylation, and inhibitors of the respective kinases inhibited shikonin-induced Nrf2 nuclear translocation.. Shikonin effectively upregulates the transcription of CYP isozymes, phase II detoxification enzymes, and phase III membrane transporters and this function is at least partially through activation of AhR and Nrf2. Moreover, Nrf2 activation is dependent on mitogen-activated protein kinases.

Topics: Animals; Anti-Inflammatory Agents; Basic Helix-Loop-Helix Transcription Factors; Biotransformation; Cells, Cultured; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Hepatocytes; JNK Mitogen-Activated Protein Kinases; Male; Membrane Transport Proteins; Naphthoquinones; NF-E2-Related Factor 2; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Primary Cell Culture; Rats, Sprague-Dawley; Receptors, Aryl Hydrocarbon; RNA, Messenger; Transcriptional Activation

Shikonin is a naphthoquinone isolated from the dried root of Lithospermum erythrorhizon, an herb used in Chinese medicine. Although several studies have indicated that shikonin exhibits antitumor activity in breast cancer, the mechanism of action remains unclear. In the present study, we performed transcriptome analysis using RNA-seq and explored the mechanism of action of shikonin in regulating the growth of different types of breast cancer cells. The IC

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Dual Specificity Phosphatase 1; Dual Specificity Phosphatase 2; Gene Expression Profiling; Humans; Lithospermum; MAP Kinase Signaling System; MCF-7 Cells; Naphthoquinones; RNA; Signal Transduction; Transcriptome

Burkitt's lymphoma (BL) is a highly aggressive malignancy molecularly characterized by deregulation of the C-MYC proto-oncogene. Recently, it has been confirmed that phosphatidylinositol-3-kinase (PI3K) pathway activation is a crucial element in the malignant transformation of the B cells in BL. Despite the better outcome of adults with BL treated with high-intensity chemotherapy regimens, the overall survival rate for patients older than 60 years remains dismal. Shikonin, a natural naphthoquinone derived from Chinese herbal medicine plant, has the potential to induce cell death in a series of human cancer. In the present study, we investigated the effect and molecular mechanisms of Shikonin in treatment with BL. Shikonin suppressed cellular proliferation and induced caspase-dependent apoptosis in BL cells. Inhibition of C-MYC and suppression of PI3K/AKT/mTOR pathway played critical roles in SHK-induced apoptosis in BL both in vitro and in vivo. Besides, Shikonin potentiated doxorubicin-induced growth inhibition and apoptosis in vitro. Furthermore, the growth of a subcutaneous xenograft tumor model of BL was significantly inhibited by shikonin. Importantly, we did not find the effect of shikonin on liver function in mice. In summary, these data suggest that shikonin may be an encouraging chemotherapeutic agent in the clinical treatment of BL.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibiotics, Antineoplastic; Apoptosis; Burkitt Lymphoma; Cell Proliferation; Doxorubicin; Drug Synergism; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Naphthoquinones; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Mas; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Shikonin, a natural naphthoquinone compound derived from the herb Lithospermum erythrorhizon, is widely used for its various pharmacological activities. However, its potential interactions with other medications by inhibiting human carboxylesterases 2 (hCE2) remain unknown. In this study, the inhibitory effects of shikonin on the activity of hCE2 in human liver microsomes are investigated by using fluorescein diacetate (FD), N-(2-butyl-1,3-dioxo-2,3-dihydro-1H-phenalen-6-yl)-2-chloroacetamide (NCEN), and CPT-11 as substrates of hCE2. The results demonstrate that shikonin significantly inhibits the activity of hCE2 when FD and NCEN are used as substrates, whereas the half inhibition concentration value of shikonin increased by 5-30 times when CPT-11 was used as the substrate. The inhibition types of shikonin against hCE2 activity reflected by 3 substrates were all best fit to noncompetitive manners. In addition, shikonin was found to distinctly suppress endogenous hCE2 activity, characterized with attenuated fluorescence. Furthermore, for drugs metabolized by hCE2 with the similar binding sites with FD or NCEN, the estimated magnitudes of area under the curve variation were approximately 9-357% in the presence of shikonin. Also, the area under the curve of CPT-11 could be increased by 1-14% following administration of shikonin. These findings have clear clinical implications for the combination of shikonin and hCE2-metabolizing prodrugs.

Topics: Carboxylesterase; Drug Combinations; Humans; Naphthoquinones; Plants, Medicinal

RIP1 and RIP3 are necroptosis initiators, but their roles in regulation of glycolysis remain elusive. In this study, we found shikonin activated RIP1 and RIP3 in glioma cells in vitro and in vivo, which was accompanied with glycolysis suppression. Further investigation revealed that shikonin-induced decreases of glucose-6-phosphate and pyruvate and downregulation of HK II and PKM2 were significantly prevented when RIP1 or RIP3 was pharmacologically inhibited or genetically knocked down with SiRNA. Moreover, shikonin also triggered accumulation of intracellular H

Topics: Animals; Cell Line, Tumor; Cysteine; Gene Expression Regulation, Neoplastic; Glioma; Glutathione; Glycolysis; Humans; Hydrogen Peroxide; Mice; Naphthoquinones; Nuclear Pore Complex Proteins; Rats; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Xenograft Model Antitumor Assays

Mutant EGFR Non-small cell lung cancer has benefit from gefitinib, but it has limited effect for wild-type EGFR tumors. Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicine, the plant Lithospermum erythrorhizon (zicao), not only can inhibit the tumor growth, but also overcome cancer drug resistance. Our aim is to investigate whether shikonin can enhance antitumor effect of gefitinib in EGFR wild-type lung cancer cells in vitro and in vivo.. CCK-8 was used to determine the proliferation of EGFR wild-type non-small cell lung cancer. Apoptosis and cell cycle were detected by flow cytometry. PKM2, STAT3, p-STAT3 and cyclinD1 were detected by Western blot. A549 tumor model was established to observe the antitumor effect of shikonin combination with gefitinib in vivo.. The results showed that combination of shikonin with gefitinib exhibited synergistic antitumor effect in vitro and in vivo. Its potential molecular mechanisms may be associated with inhibition of PKM2/STAT3/cyclinD1.. These results provide a promising therapeutic approach for the treatment of wild-type EGFR non-small cell lung cancer.

Topics: A549 Cells; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Synergism; ErbB Receptors; Gefitinib; Humans; Immunohistochemistry; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Naphthoquinones; Quinazolines; Signal Transduction; Sincalide; STAT3 Transcription Factor; Thyroid Hormone-Binding Proteins; Thyroid Hormones

The Proteins involved in the chemical modification of lysine residues in histone, is currently being excessively focused as the therapeutic target for the treatment of cell related diseases like cancer. Among these proteins, the epigenetic reader, CREB-binding protein (CREBBP) bromodomain is one of the most prominent targets for effective anticancer drug design, which is responsible for the reorganization of acetylated histone lysine residues. Therefore, this study employed an integrative approach of structure based drug design, in combination with Molecular Dynamics (MD) and QM/MM study to identify as well as to describe the binding mechanism of two shikonin derivatives, acetylshikonin and propionylshikonin as inhibitors of CREBBP bromodomain. Here induced fit docking strategy was employed to explore the important intrinsic interactions of ligands with CREBBP bromodomain, consistently molecular dynamics simulation with two different methods and binding energy calculations by MM-GBSA and MM-PBSA were adopted to determine the stability of intermolecular interactions between protein and ligands. The results showed that both these derivatives made direct contacts with the important conserved residues of the active site, where propionylshikonin demonstrated stronger binding and stability than acetylshikonin, according to molecular dynamics simulation and binding free energy calculations. Further, QM/MM energy calculation was employed to study the chemical reactivity of the propionylshikonin and also to describe the mechanism of non bonded interactions between the propionylshikonin and CREBBP bromodomain. Though this study demands in vitro and in vivo experiments to evaluate the efficiency of the compound, these insights would assist to design more potent CREBBP bromodomain inhibitor, guiding the site of modification of propionylshikonin moiety for designing selective inhibitors.

Topics: Binding Sites; Catalytic Domain; CREB-Binding Protein; Drug Design; Ligands; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Naphthoquinones; Protein Binding; Protein Interaction Domains and Motifs; Quantitative Structure-Activity Relationship; Quantum Theory; Thermodynamics

Myocardial ischemic/reperfusion (I/R) injury often leads to irreversible myocardial cell death and even heart failure, with limited therapeutic possibilities. In the present study, we evaluated the protective effects of shikonin (SHK) against hypoxia/reoxygenation (H/R)-induced cardiomyocyte damage and explored the underlying mechanisms. H9C2 cardiomyocytes were pretreated with different doses of SHK prior to H/R exposure. We observed that SHK pretreatment significantly increased cell viability, attenuated LDH release, and suppressed cardiomyocyte apoptosis induced by H/R exposure. SHK pretreatment also restored the loss of mitochondrial membrane potential (MMP) and cytochrome c release. In addition, SHK significantly enhanced the phosphorylation of Akt and GSK-3β in H/R-treated H9C2 cells. These protective effects of SHK were partially reversed by LY294002, a specific PI3K/Akt inhibitor. Therefore, our findings suggested that SHK might be a promising agent for myocardial I/R injury, and PI3K/Akt signaling plays a crucial role during this process.

Topics: Animals; Apoptosis; Cardiotonic Agents; Cell Line; Cell Survival; Chromones; Glycogen Synthase Kinase 3 beta; Hypoxia; Membrane Potential, Mitochondrial; Mitochondria; Morpholines; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Naphthoquinones; Phosphatidylinositol 3-Kinases; Phosphorylation; Protective Agents; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm. Tyrosine kinase inhibitors (TKIs) are commonly used to treat CML; however, drug resistance of CML cells to TKIs has limited their clinical application. Shikonin, a traditional Chinese herb, has long been used to treat leukemia in China, but the roles and related molecular mechanisms of shikonin treatment in CML remain unclear. Here, we aimed to evaluate the effects of shikonin on the proliferation, apoptosis, and migration of K562 cells, a CML cell line.. Firstly, K562 cell proliferation and apoptosis were tested by CCK8 assay and flow cytometry with Annexin V-FITC/PI staining. Cell migration was measured by Transwell migration assay. In addition, western blot was performed to determine the proteins (PI3K, Bax, Bcl-2, cleaved caspase-3, PTEN, p-AKT, AKT, CXCR4, SDF-1, CD44) involved in the mechanism of action of shikonin. Finally, neutrophils from peripheral blood of CML patients were obtained, and cell proliferation and apoptosis were tested by CCK8 assay and flow cytometry.. Shikonin reduced the proliferation of K562 cells in a time- and dose-dependent manner and promoted the apoptosis of K562 cells. Moreover, shikonin increased the PTEN level and inactivated the PI3K/AKT signaling pathway, subsequently upregulating BAX in K562 cells. In addition, shikonin could block K562 cell migration via the CXCR4/SDF-1 axis. Finally, shikonin significantly inhibited the proliferation and promoted the apoptosis of neutrophils from CML patients.. These results demonstrated that shikonin inhibits CML proliferation and migration and induces apoptosis by the PTEN/PI3K/AKT pathway, revealing the effects of shikonin therapy on CML.

Topics: Apoptosis; Cell Proliferation; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction

Glycation and advanced glycation end products (AGE) damage skin which is compounded by AGE-induced oxidative stress and inflammation. Lip and facial skin could be susceptible to glycation damage as they are chronically stressed. As Gromwell (Lithospermum erythrorhizon) root (GR) has an extensive traditional medicine history that includes providing multiple skin benefits, our objective was to determine whether GR extract and its base naphthoquinone, shikonin, might protect skin by inhibiting glycation, increasing oxidative defenses, suppressing inflammatory responses and offering ultraviolet (UV) absorptive potential in lip and facial cosmetic matrices. We show GR extract and shikonin dose-dependently inhibited glycation and enhanced oxidative defenses through nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element activation. Inflammatory targets, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and tumor necrosis factor alpha, were suppressed by GR extract and shikonin. Glyoxalase 1 (GLO1) and glutathione synthesis genes were significantly upregulated by GR extract and shikonin. GR extract boosted higher wavelength UV absorption in select cosmetic matrices. Rationale for the use of GR extract and shikonin are supported by our research. By inhibiting glycation, modulating oxidative stress, suppressing inflammation and UV-absorptive properties, GR extract and shikonin potentially offer multiple skin benefits.

Topics: Absorption, Radiation; Anti-Inflammatory Agents, Non-Steroidal; Cosmetics; Glutathione; Glycation End Products, Advanced; Hep G2 Cells; Humans; Inflammation; Lactoylglutathione Lyase; Lithospermum; Naphthoquinones; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Plant Extracts; Plant Roots; Tumor Necrosis Factor-alpha; Ultraviolet Rays; Up-Regulation

Drug delivery is considered a mature scientific and technological platform for producing innovative medicines with nanosystems composed of intelligent bio-materials that carry active pharmaceutical ingredients forming advanced drug delivery nanosystems (aDDnSs). Shikonin and its enantiomer alkannin are natural products that have been extensively studied in vitro and in vivo for, among others, their antitumor activity, and various efforts have been made to prepare shikonin-loaded drug delivery systems. This study is focused on chimeric aDDnSs and specifically on liposomal formulations combining three lipids (egg-phosphatidylcholine; dipalmitoyl phosphatidylcholine; and distearoyl phosphatidylcholine) and a hyperbranched polymer (PFH-64-OH). Furthermore, PEGylated liposomal formulations of all samples were also prepared. Calorimetric techniques and electron paramagnetic resonance were used to explore and evaluate the interactions and stability of the liposomal formulations, showing that the presence of hyperbranched polymers promote the overall stability of the chimeric aDDnSs based on the drug release profile enhancement. Furthermore, results showed that polyethylene glycol enhances drug stabilization inside the liposomes, forming a stable and promising carrier for shikonin with improved characteristics.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Antineoplastic Agents; Calorimetry; Drug Carriers; Electron Spin Resonance Spectroscopy; Liposomes; Naphthoquinones; Particle Size; Phosphatidylcholines; Polyethylene Glycols; Static Electricity

The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is of importance in pulmonary vascular remodeling. Shikonin, a naphthoquinone compound extracted from the Chinese medicinal herb Lithospermum erythrorhizon, inhibits the proliferation of rat smooth muscle cells (SMCs). The present study was designed to investigate the effects of shikonin on the proliferation of rat PASMCs and the possible mechanisms involved. Rat PASMCs were cultured under the following five treatment conditions: Normal control; hypoxia for 24 h; hypoxia + 1 µM shikonin for 24 h; hypoxia + 2 µM shikonin for 24 h; and hypoxia + 4 µM shikonin for 24 h. The viability of PASMCs was measured using the Cell Counting Kit‑8 assay, the mRNA expression of nestin (NES) in each group was measured by reverse transcription‑polymerase chain reaction and the protein expression of NES was measured by western blotting. The proliferation of hypoxic PASMCs transfected with NES‑specific small interfering (si)RNA decreased compared with the non‑transfected group. These results indicated that hypoxia induced the proliferation of PASMCs through the enhancement of NES expression. The treatment of hypoxic PASMCs with shikonin resulted in a significant downregulation of NES expression and the inhibition of PASMC proliferation.

Topics: Animals; Cell Hypoxia; Cell Proliferation; Cells, Cultured; Gene Expression Regulation; Hypoxia; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthoquinones; Nestin; Pulmonary Artery; Rats; RNA, Small Interfering

We evaluated the skin sensitizing potential of 10 natural organic chemicals, or their derivatives, which are included in foods and/or skin products, using a modified local lymph node assay (LLNA), with an elicitation phase (LLNA:DAE). The following compounds were tested: carminic acid, esculetin, 4-methyl esculetin, coumarin, quercetin, curcumin, naringenin, chlorogenic acid, isoscopoletin, and shikonin. Esculetin, 4-methyl esculetin, isoscopoletin, and shikonin yielded positive results. In particular, shikonin at a very low concentration (0.05%) induced an elicitation response. In conclusion, four of the 10 natural organic chemicals tested had a skin sensitization potential, with shikonin producing serious reaction even at a very low concentration.

Topics: Animals; Carmine; Cosmetics; Coumarins; Curcumin; Dose-Response Relationship, Drug; Female; Food Analysis; Local Lymph Node Assay; Mice, Inbred CBA; Naphthoquinones; Quercetin; Skin; Skin Irritancy Tests; Umbelliferones

As patients with non‑small cell lung cancer (NSCLC) and wild‑type epidermal growth factor receptor (EGFR) are resistant to treatment with erlotinib or gefitinib, potential chemosensitizers are required to potentiate wild‑type EGFR NSCLC cells to erlotinib/gefitinib treatment. The present study reported that shikonin could sensitize the anticancer activity of erlotinib/gefitinib in wild‑type EGFR NSCLC cells. Furthermore, shikonin could potentiate mitochondrial‑mediated apoptosis induced by erlotinib/gefitinib in wild‑type EGFR NSCLC cells. In addition, the present study demonstrated that shikonin could induce apoptosis by activating reactive oxygen species (ROS)‑mediated endoplasmic reticulum (ER) stress, and that erlotinib/gefitinib may also induce ER stress in wild‑type EGFR NSCLC cells; however, shikonin plus erlotinib/gefitinib was more effective in activating ER stress than either agent alone. This indicated that ROS‑mediated ER stress may be associated with enhanced mitochondrial apoptosis induced by shikonin plus erlotinib/gefitinib. In addition, shikonin may promote the transition of cytoprotective ER stress‑inducing EGFR‑tyrosine kinase inhibitor tolerance to apoptosis‑promoting ER stress. Furthermore, shikonin may enhance the anti‑NSCLC activity of erlotinib/gefitinib in vivo. The data of the present study indicated that shikonin may be a potential sensitizer to enhance the anti‑cancer efficacy of erlotinib/gefitinib in wild‑type EGFR NSCLC cells resistant to erlotinib/gefitinib treatment.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Lithospermum; Lung Neoplasms; Mice, Nude; Naphthoquinones; Protein Kinase Inhibitors; Reactive Oxygen Species

Previous studies have shown that shikonin(SHI), the bioactive naphthoquinone constituent extracted from Chinese herb Lithospermum Erythrorhizon, possesses the potential to confront inflammation, and has little concerns towards drug residues comparing with antibiotics. While mastitis in dairy industry always trigger great harm to milk yields, effects of SHI on lipopolysaccharides (LPS)-induced mastitis should be measured. Here, we demonstrate anti-inflammatory effects of SHI on LPS-challenged mastitis and elucidate the potential signaling pathway both in vivo and in vitro. As a result, SHI administration mice significantly suffered less impairment of mammary gland and less recruitment of neutrophils than LPS administration mice. SHI significantly suppressed the expression of p-IκBα and p-p65, which are the critical proteins functioning in NF-kB signaling pathway. qPCR results indicate decreasing level of upstream pro-inflammatory cytokines in tissues, such as TNF-α, IL-1β, and IL-6. The results are corresponding with the results in vitro, suggesting the potential usage of SHI as a therapeutic medicine in mastitis.

Topics: Animals; Anti-Inflammatory Agents; Cell Line, Tumor; Cytokines; Female; Gene Expression; Lipopolysaccharides; Mammary Glands, Animal; Mastitis; Mice; Molecular Structure; Naphthoquinones; NF-kappa B; Peroxidase; Signal Transduction

Shift metabolism profile from mitochondrial oxidative phosphorylation to aerobic glycolysis (Warburg effect) is a key for tumor cell growth and metastasis. Therefore, suppressing the tumor aerobic glycolysis shows a great promise in anti-tumor therapy. In the present study, we study the role of shikonin, a naphthoquinone isolated from the traditional Chinese medicine Lithospermum, in inhibiting tumor aerobic glycolysis and thus tumor growth. We found that shikonin dose-dependently inhibited glucose uptake and lactate production in Lewis lung carcinoma (LLC) and B16 melanoma cells, confirming the inhibitory effect of shikonin on tumor aerobic glycolysis. Treatment of shikonin also decreased tumor cell ATP production. Furthermore, pyruvate kinase M2 (PKM2) inhibitor or activator respectively altered the effect of shikonin on tumor cell aerobic glycolysis, suggesting that suppression of cell aerobic glycolysis by shikonin is through decreasing PKM2 activity. Western blot analysis confirmed that shikonin treatment reduced tumor cell PKM2 phosphorylation though did not reduce total cellular PKM2 level. In vitro assay also showed that shikonin treatment significantly promoted tumor cell apoptosis compared to untreated control cells. Finally, when mice implanted with B16 cells were administered with shikonin or control vehicle, only shikonin treatment significantly decreased B16 tumor cell growth. In conclusion, this study demonstrates that shikonin inhibits tumor growth in mice by suppressing PKM2-mediated aerobic glycolysis.

Topics: Adenosine Triphosphate; Aerobiosis; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Enzyme Inhibitors; Glycolysis; Male; Melanoma, Experimental; Mice; Mice, Nude; Mice, SCID; Molecular Targeted Therapy; Naphthoquinones; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Pyruvate Kinase; Stomach Neoplasms

Hyperthermia (HT) has been used widely for cancer therapy, and the development of modern devices has made it more efficient. Shikonin (SHK) is a natural naphthoquinone derivative from a Chinese herb. Although the anticancer properties of SHK are evident, the underlying molecular mechanisms are not fully understood.. In this study, the effects of combining low doses of SHK with mild HT were investigated in the U937 cell line.. The cells were subjected to HT at 44°C for 10 min with or without SHK pretreatment, and parameters reflecting apoptosis, ROS generation and intracellular calcium elevation were evaluated by using DNA fragmentation, flow cytometry, and western blot analyses.. SHK 0.5 µM significantly enhanced HT-induced apoptosis as indicated by DNA fragmentation and caspase-3 activation with increased generation of ROS and elevation of intracellular calcium. The combined treatment also synergistically activated proapoptotic proteins and inactivated anti-apoptotic proteins. Furthermore, the phosphorylation of JNK and PKC- δ and the dephosphorylation of ERK and AKT were the upstream effects that may have compounded the induction of apoptosis. The modulatory effects of HT and SHK were abrogated with the employment of NAC and JNK-IN-8 by inactivating the MAPK pathway and cleavage of caspase-3. Intracellular calcium was also elevated and was found to be responsible for the induction of cell death evident by the DNA fragmentation with or without the employment of BAPTA-AM.. Conclusively, this study provides persuasive evidence that SHK in combination with HT is a propitious therapeutic way for augmentation of apoptosis and hence suggest a novel strategy for treating cancers.

Topics: Apoptosis; Humans; Hyperthermia, Induced; Naphthoquinones; Neoplasm Proteins; Neoplasms; Oxidative Stress; Reactive Oxygen Species; U937 Cells

Despite much research in the last centuries, treatment of malignant melanoma is still challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate. Therefore, the discovery of novel drug leads is an important goal. In a previous study, we have isolated several shikonin derivatives from the roots of

Topics: Apoptosis; Boraginaceae; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Membrane; Cell Proliferation; DNA Breaks, Double-Stranded; Histones; Humans; Melanoma; Naphthoquinones; Phosphorylation; Plant Roots; Skin

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, promotes cancer growth and metastasis. Finding natural compounds with anti-lymphangiogenic activity will be useful for preventive treatment of lymphatic metastasis. Shikonin, an ingredient of a traditional Japanese and Chinese medicinal herb Lithospermum erythrorhizon, has been widely used in several pharmaceutical and cosmetic preparations, as well as in food colorants. Shikonin has been reported to inhibit lymphangiogenesis in vitro, but the mechanism of inhibition has not been determined. The aim of this study is to investigate the mechanism of anti-lymphangiogenesis of shikonin in primary human lymphatic endothelial cells (HMVEC-dLy). Shikonin, at non-toxic concentrations, significantly inhibited cord formation ability of lymphatic endothelial cells in a dose- and time-dependent manner. Western blotting analysis showed that shikonin decreased nuclear factor-kappaB (NF-κB) activation, as indicated by phosphorylation and nuclear translocation of NF-κB p65, and also reduced both mRNA and protein levels of hypoxia-inducible factor-1 (HIF-1)α. Use of an NF-κB inhibitor (BAY 11-7085) and HIF-1α small interfering RNA (siRNA) transfection revealed that NF-κB activation was upstream of HIF-1α expression, which controls cord formation by HMVEC-dLy. In addition, the reduction of vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3) mRNA levels were also found in HMVEC-dLy that treated with shikonin. In conclusion, shikonin inhibits lymphangiogenesis in vitro by interfering the NF-κB/HIF-1α pathway and involves in suppression of VEGF-C and VEGFR-3 mRNA expression.

Topics: Drugs, Chinese Herbal; Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lithospermum; Lymphangiogenesis; Lymphatic Metastasis; Naphthoquinones; NF-kappa B; Phytotherapy; RNA, Messenger; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factor Receptor-3

Shikonin (SHI), an active component extracted from Radix Arnebiae, has been reported to possess anti-inflammatory properties in various cells. However, its effect on lipopolysaccharide (LPS)-stimulated human periodontal ligament cells (hPDLCs) is unknown.. To investigate the effects of SHI on the expression of inflammatory related cytokines in LPS-stimulated hPDLCs.. The effects of SHI (0.125, 0.25, 0.5, 1, and 2 μg/mL) on hPDLCs proliferation for 1, 3 and 7 days were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of interleukin-1 (IL-1), IL-6, tumor necrosis factor-α (TNF-α), matrix metalloproteinase-2 (MMP-2), MMP-9 and cyclooxygenase-2 (COX-2) were detected in hPDLCs following SHI treatment (0.25 and 0.5 μg/mL) using Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). The signaling pathways triggered by SHI in hPDLC were evaluated using western blotting.. LD50 of SHI is 1.7 μg/mL (day 1) and 1.1 μg/mL (day 3 and 7) in hPDLCs. No morphological changes were observed when hPDLCs were treated with LPS only (1 μg/mL) or LPS with SHI (0.25 and 0.5 μg/mL). Data from qRT-PCR suggests that SHI attenuates LPS-induced increases of IL-1, IL-6, TNF-α, MMP-2, MMP-9 and COX-2 in hPDLCs. Down-regulation of phosphorylated extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB), and up-regulation of I-κB, were observed in LPS-stimulated hPDLCs after exposed to SHI at 0.25 or 0.5 μg/mL.. SHI possesses anti-inflammatory effects in LPS-stimulated hPDLCs via phospho-ERK and NF-κB/I-κB signaling pathways; this suggests that SHI may hold potential as an anti-inflammatory agent against periodontitis.

Topics: Anti-Inflammatory Agents; Cell Line; Cell Proliferation; Cyclooxygenase 2; Cytokines; Humans; Lipopolysaccharides; MAP Kinase Signaling System; Matrix Metalloproteinases; Naphthoquinones; Periodontal Ligament; Periodontitis; Phosphorylation

Topics: Afatinib; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Lithospermum; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Cisplatin-based chemotherapy often results in the development of chemo-resistance when used to treat bladder cancer (BC), which is difficult to overcome. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC, and contributes to the cisplatin resistance in BC. However, the underlying mechanisms remain unclear. In this study, we also found that the expression level of PKM2 is also higher in cisplatin resistant BC cells and tumors. Down-regulation of PKM2 by siRNA or inhibition of PKM2 by shikonin re-sensitized the cisplatin resistant T24 cells. Shikonin and cisplatin together exhibit significantly greater killing effects than when used alone. Interestingly, we found shikonin kills the T24 cisplatin resistant cells by inducing necroptosis, as the cell death could not inhibited by apoptosis inhibitor, z-VAD, but compromised by RIP3 inhibitor, GSK872, or RIP3 siRNA. In contrast, shikonin induced apoptosis in T24 parental cells. We further investigate the underlying mechanism, and found that the dysregulation of Bcl-2 family proteins, including Bcl-2, PUMA, Bax, play an important role in deciding that shikonin kills the BC cells by necroptosis or apoptosis. Collectively, our results suggested that inducing necroptosis is an alternative way to overcome the apoptosis resistant in BC therapy, and orchestrating the regulation of Bcl-2, PUMA, and Bax in BC cisplatin resistant cells may improve the therapy effect of cisplatin in BC tumor.

Topics: Aged; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Carrier Proteins; Cell Line, Tumor; Cisplatin; Female; Humans; In Vitro Techniques; Male; Membrane Proteins; Middle Aged; Naphthoquinones; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Urinary Bladder Neoplasms

TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, can selectively kill cancer cells with little or no cytotoxicity toward normal human cells and is regarded as a potential relatively safe antitumor drug. However, some cancer cells are resistant to TRAIL-induced apoptosis. Thus, reagents that potentiate TRAIL-induced cytotoxicity are needed. Herein, we investigated whether shikonin, a natural compound from the root of Lithospermum erythrorhizon, can sensitize TRAIL-resistant cells to TRAIL-induced cytotoxicity.. The viability of A549 cells, which were resistant to TRAIL, was significantly decreased after treatment with TRAIL followed by shikonin. The underlying mechanisms by which shikonin sensitizes cells to TRAIL-induced cytotoxicity were also examined. Combined treatment with shikonin and TRAIL activated the caspase and JNK pathways, inhibited the STAT3 and AKT pathways, downregulated the expression of Mcl-1, Bcl-2, Bcl-xL, c-FLIP and XIAP and upregulated the expression of Bid.. In conclusion, the results indicated that shikonin sensitized resistant cancer cells to TRAIL-induced cytotoxicity via the modulation of the JNK, STAT3 and AKT pathways, the downregulation of antiapoptotic proteins and the upregulation of proapoptotic proteins.

Topics: A549 Cells; Apoptosis; Cell Death; Drug Synergism; HEK293 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Naphthoquinones; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; TNF-Related Apoptosis-Inducing Ligand

Acetaminophen (APAP) overdose-induced acute liver damage is mostly due to overwhelmingly increased oxidative stress. Nuclear factor-erythroid 2-related factor2 (Nrf2) plays an important role in alleviating APAP hepatic toxicity. Shikonin (SHK) enhances Nrf2 in multiple lines of normal cells. Nevertheless, whether SHK protects against APAP-induced liver toxicity remains undefined. This study found SHK defended APAP-induced liver toxicity, as well as reversed the levels of serum alanine/aspartate aminotransferases (ALT/AST), liver myeloperoxidase (MPO) activity, and reactive oxygen species (ROS), while it enhanced the liver glutathione (GSH) level in APAP-treated mice. SHK rescued the cell viability and GSH depletion, but neutralized oxidative stress in APAP-treated human normal liver L-02 cells. Mechanically, SHK increased Nrf2 expression in the exposure of APAP at the protein level but not at the mRNA level. Inhibition of Nrf2 blocked the SHK effect in APAP-treated hepatocytes. Furthermore, SHK improved Nrf2 stability through stimulating PI3K/Akt pathway, thus inhibiting GSK-3β. In vivo studies confirmed the close correlation of liver protection of SHK against APAP and Akt/GSK-3β/Nrf2 pathway. In conclusion, this study reveals that SHK prevents APAP hepatotoxicity by upregulation of Nrf2 via PI3K/Akt/GSK-3β pathway. Therefore, SHK may be a promising candidate against APAP-induced liver injury.

Topics: Acetaminophen; Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomarkers; Biopsy; Cell Line; Chemical and Drug Induced Liver Injury; Gene Expression; Glycogen Synthase Kinase 3 beta; Hepatocytes; Male; Mice; Naphthoquinones; NF-E2-Related Factor 2; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

Hepatic ischemia/reperfusion (I/R) injury, which can result in severe liver injury and dysfunction, occurs in a variety of conditions such as liver transplantation, shock, and trauma. Cell death in hepatic I/R injury has been linked to apoptosis and autophagy. Shikonin plays a significant protective role in ischemia/reperfusion injury. The purpose of the present study was to investigate the protective effect of shikonin on hepatic I/R injury and explore the underlying mechanism. Mice were subjected to segmental (70%) hepatic warm ischemia to induce hepatic I/R injury. Two doses of shikonin (7.5 and 12.5 mg/kg) were administered 2 h before surgery. Balb/c mice were randomly divided into four groups: normal control, I/R, and shikonin preconditioning at two doses (7.5 and 12.5 mg/kg). The serum and liver tissues were collected at three time points (3, 6, and 24 h). Shikonin significantly reduced serum AST and ALT levels and improved pathological features. Shikonin affected the expression of Bcl-2, Bax, caspase 3, caspase 9, Beclin-1, and LC3, and upregulated PI3K and p-Akt compared with the levels in the I/R group. Shikonin attenuated hepatic I/R injury by inhibiting apoptosis and autophagy through a mechanism involving the activation of PI3K/Akt signaling.

Topics: Animals; Apoptosis; Autophagy; Cytokines; Dimethyl Sulfoxide; Enzyme Activation; Hepatocytes; Inflammation Mediators; Liver; Liver Function Tests; Male; Mice, Inbred BALB C; Models, Biological; Naphthoquinones; Phosphatidylinositol 3-Kinases; Protective Agents; Proto-Oncogene Proteins c-akt; Reperfusion Injury; Signal Transduction

Chemoresistance to cisplatin is a principal cause of treatment failure and mortality of advanced bladder cancer (BC). The underlying mechanisms remain unclear, which hinders the development of preventive strategies. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC. This study explores the role of PKM2 in chemoresistance and whether inhibiting PKM2 augments the chemosensitivity to cisplatin and reduces BC growth and progression. We found that Shikonin binds PKM2 and inhibits BC cell survival in a dose-dependent but pyruvate kinase activity-independent manner. Down-regulation of PKM2 by shRNA blunts cellular responses to shikonin but enhances the responses to cisplatin. Shikonin and cisplatin together exhibit significantly greater inhibition of proliferation and apoptosis than when used alone. Induced cisplatin-resistance is strongly associated with PKM2 overexpression, and cisplatin-resistant cells respond sensitively to shikonin. In syngeneic mice, shikonin and cisplatin together, but not as single-agents, markedly reduces BC growth and metastasis. Based on these data, we conclude that PKM2 overexpression is a key mechanism of chemoresistance of advanced BC to cisplatin. Inhibition of PKM2 via RNAi or chemical inhibitors may be a highly effective approach to overcome chemoresistance and improve the outcome of advanced BC.

Topics: Actins; Aged, 80 and over; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Polymerization; Protein Kinase Inhibitors; Pyruvate Kinase; RNA, Small Interfering; Up-Regulation; Urinary Bladder Neoplasms

Streptococcus pneumoniae (S. pneumoniae) is a common pathogen that can cause severe infections in humans. Pneumolysin (PLY) is an important virulence trait of S. pneumoniae and has cytotoxicity, genotoxicity and pro-inflammatory activity; it is essential for the pathogenesis of S. pneumoniae pneumonia and is an anti-virulence target of small molecule drug development. The treatment options for this microbe were limit due to the ubiquitous antibiotic resistance; therefore, new drugs and treatment strategies are needed.. Shikonin was selected by drug screening based on haemolysis assays, and its mechanism of suppressing PLY toxicity was determined by oligomerization assay. Meanwhile, the in vitro cell viability assays and in vivo experiments were performed to explore the capability of shikonin to protect cells and tissue from S. pneumoniae-mediated damage.. Shikonin was found to significantly decrease PLY-induced haemolytic activity, cytotoxicity and genotoxicity via lessening the formation of oligomers; moreover, the agent can reduce the mortality of mice caused by lethal pneumonia and mitigate the injury of target organs as well.. We suggest that shikonin could be a potent candidate for a novel therapeutic or auxiliary substance in the treatment of infections encountering insufficient vaccines and antimicrobial resistance to traditional antibiotics.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Cell Survival; Drug Design; Female; Hemolysis; Humans; Mice; Mice, Inbred C57BL; Naphthoquinones; Pneumonia, Pneumococcal; Streptococcus pneumoniae; Streptolysins

Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells.. The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry.. We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling.. From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

Topics: Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dacarbazine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Glioblastoma; Humans; Naphthoquinones; Temozolomide

Epithelial-to-mesenchymal transition (EMT) is a major process to regulate cell migration and invasion. Inhibition of epidermal growth factor receptor (EGFR)-mediated EMT by tyrosine kinase inhibitors (TKIs) is a strategy to prevent lung cancer invasion. However, drug resistance is emerged and accelerated invasion through other signaling bypassing EGFR after TKIs therapy. c-Met signaling pathway is highly activated in EGFR-mutated lung cancer cells. Targeting c-Met signaling pathway may be a strategy to suppress EGFR-independent migration and invasion for lung cancer therapy. Therefore, we examined the anti-migration and anti-invasion abilities of shikonin, an active compound from Lithospermum erythrorhizon, in highly and ligand-induced c-Met activation lung cancer cells. Our results revealed that cell viability and cell cycle progression did not change under 1 μM of shikoinin treatment in highly c-Met expressive HCC827 lung cancer cells. Endogenous c-Met activation was dose-dependently inhibited and the migration and invasion activity of HCC827 cells were suppressed by shikonin treatment. Induction of E-cadherin expression and inhibition of vimentin, slug, and snail expression by shikonin was through c-Met-mediated PI3K/Akt and ERK signaling suppression. Furthermore, hepatocyte growth factor (HGF)-induced migration, invasion and EMT marker change were reversed by shikonin in low c-Met expressive A549 lung cancer cells. Inhibition of HGF-induced c-Met, PI3K/Akt and MEK/ERK activation were observed in shikonin-treated cells. Co-treatment of PI3K/Akt inhibitor or ERK inhibitor with shikonin enhanced shikonin-reversed HGF-regulated EMT marker expression. Taken together, the results suggested that the anti-migration and anti-invasion activities of shikonin was through c-Met inhibition and following by EMT suppression in lung cancer. J. Cell. Biochem. 118: 4639-4651, 2017. © 2017 Wiley Periodicals, Inc.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; MAP Kinase Signaling System; Naphthoquinones; Neoplasm Invasiveness; Proto-Oncogene Proteins c-met

Shikonin is a naphthoquinone extracted from the root of Lithospermum erythrorhizon, and has been reported to suppress allergic airway inflammation in mice. However, the underlying mechanisms are unclear and need to be further elucidated. In this study, shikonin (0.5, 2 or 4mg/kg) was given intraperitoneally to ovalbumin (OVA)-challenged BALB/c mice. We found that the pathological airway remodeling of asthmatic mice was alleviated by shikonin, and the infiltrated inflammatory cells and collagen deposition in their lungs were reduced. Furthermore, the abnormal activation of extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) pathway and the elevation of matrix metalloproteinase 9 (MMP9) in the lung of asthmatic mice were suppressed by shikonin. The inactivation of NF-κB by shikonin was at least in part via inhibiting IκBα activation. In vitro, the treatment of shikonin inhibited the platelet-derived growth factor (PDGF)-induced proliferation of primary airway smooth muscle cells (ASMCs), and induced a G0/G1 arrest in these cells. In addition, ASMCs exposed to PDGF acquired an enhanced migratory ability, and the activities of MMP9 and matrix metalloproteinase 2 (MMP2) and expression of MMP9 of these cells were significantly up-regulated. These PDGF-induced alterations were also inhibited by shikonin. The inhibitory effects of shikonin on the proliferation and migration of ASMCs were comparable to pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-κB pathway. In conclusion, the present study sheds lights on how shikonin alleviates allergic airway remodeling.

Topics: Airway Remodeling; Animals; Anti-Inflammatory Agents; Asthma; Bronchoalveolar Lavage Fluid; Cell Movement; Cells, Cultured; Lung; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice, Inbred BALB C; Myocytes, Smooth Muscle; Naphthoquinones; NF-kappa B; Platelet-Derived Growth Factor; Rats, Sprague-Dawley; Signal Transduction

Shikonin, a natural small agent, has shown inhibitory effect in many kinds of cells, which increases intracellular reactive oxygen species (ROS) level and causes mitochondrial injury. In this study, shikonin showed good inhibitory effect on nasopharyngeal carcinoma CNE-2Z cells in vivo and vitro. The results presented here revealed that ROS levels increased markly after shikonin treated. The electron microscopy displays the change in ultrastructure of CNE-2Z cells after treatment for shikonin, which indicated that shikonin induced necroptosis. Shikonin-induced cell death was inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the activity was unaffected by the caspase inhibitor z-VAD-fmk. Furthermore, we have demonstrated that the activation of receptor-interacting kinase (RIP) led to necroptosis. Meanwhile, shikonin also significantly inhibited tumor growth in a CNE-2Z xenograft mouse model. Taken together, shikonin induced CNE-2Z cells death by producing ROS as a necroptosis inducer. It could serve as a new therapeutic agent for treating CNE-2Z cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma; Cell Line, Tumor; Heterografts; Humans; Mice; Mice, Nude; Naphthoquinones; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Necrosis; Reactive Oxygen Species

Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action.. Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo.. Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin.. Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

Topics: A549 Cells; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 8; Cell Line, Tumor; Gene Silencing; Humans; Imidazoles; Indoles; Lithospermum; Lung Neoplasms; Macrolides; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Necrosis; Neoplasm Transplantation; RNA, Small Interfering; X-Ray Microtomography

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Magnetic Resonance Spectroscopy; Membrane Potential, Mitochondrial; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Structure; Naphthoquinones; Protein Transport; src Homology Domains; STAT3 Transcription Factor; Structure-Activity Relationship

The Chinese traditional medicine Shikonin is an ideal drug due to its multiple targets to tumor cells. But in clinics, improving its aqueous solubility and tumor accumulation is still a challenge. Herein, a copolymer with tunable poly(N-isopropylacrymaide) and polylactic acid block lengths is designed, synthesized, and characterized in nuclear magnetic resonance. The corresponding thermosensitive nanomicelle (TN) with well-defined core-shell structure is then assembled in an aqueous solution. For promoting the therapeutic index, the physical-chemistry properties of TNs including narrow size, low critical micellar concentration, high serum stability, tunable volume phase transition temperature (VPTT), high drug-loading capacity, and temperature-controlled drug release are systematically investigated and regulated through the fine self-assembly. The shikonin is then entrapped in a degradable inner core resulting in a shikonin-loaded thermosensitive nanomicelle (STN) with a VPTT of ~40°C. Compared with small-molecular shikonin, the in vitro cellular internalization and cytotoxicity of STN against breast cancer cells (Michigan Cancer Foundation-7) are obviously enhanced. In addition, the therapeutic effect is further enhanced by the programmed cell death (PCD) specifically evoked by shikonin. Interestingly, both the proliferation inhibition and PCD are synergistically promoted as T > VPTT, namely the temperature-regulated passive targeting. Consequently, as intravenous injection is administered to the BALB/c nude mice bearing breast cancer, the intratumor accumulation of STNs is significantly increased as T > VPTT, which is regulated by the in-house developed heating device. The in vivo antitumor assays against breast cancer further confirm the synergistically enhanced therapeutic efficiency. The findings of this study indicate that STN is a potential effective nanoformulation in clinical cancer therapy.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Delayed-Action Preparations; Drugs, Chinese Herbal; Female; Humans; Hyperthermia, Induced; MCF-7 Cells; Mice, Inbred BALB C; Micelles; Nanostructures; Naphthoquinones; Polyesters; Polymers; Solubility; Temperature; Tissue Distribution; Xenograft Model Antitumor Assays

There is an increasing need for the discovery of reliable and eco-friendly pesticides and natural plant-derived products may play a crucial role as source of new active compounds. In this research, a lipophilic extract of

Topics: Acaricides; Animals; Boraginaceae; Naphthoquinones; Plant Extracts; Plant Roots; Tetranychidae

Osteosarcoma is the most common primary malignant bone tumor. Cancer cells employ a host of mechanisms to develop resistance to adriamycin (ADM) or other chemotherapeutic drugs. Shikonin (SK), an active constituent extracted from a Chinese medicinal herb, has been shown to cooperate with ADM in the treatment of osteosarcoma and certain other types of cancer by contributing to the response rate of chemotherapy and the side effects. The aim of the present study was to investigate the role and underlying mechanism of SK in chemotherapy for osteosarcoma. In the present study, a CCK-8 assay was performed to assess cell survival rate in vitro. Western blot analysis was performed to determine the expression levels of B‑cell lymphoma 2‑associated X protein (Bax), caspase‑3, caspase‑8, and poly (ADP‑ribose) polymerase (PARP). Flow cytometry was used to analyze cell cycle and cell death. The survival rate of cells decreased significantly in a dose‑ and time‑dependent manner when treated with a combination of SK and ADM. Western blot analysis revealed increased expression levels of Bax, caspase‑3, caspase‑8 and PARP in U2OS and MG63 cells 48 h following treatment with SK and ADM. Flow cytometric analysis showed that the combined treatment of SK and ADM significantly induced apoptosis in the osteosarcoma cells. Taken together SK cooperated with ADM to promote apoptosis, possibly by inducing caspase‑3‑ and caspase‑8‑dependent apoptosis. SK may be a potential enhancer in the treatment of drug‑resistant primary osteosarcoma.

Topics: Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cell Survival; Doxorubicin; Drug Synergism; Humans; Naphthoquinones; Osteosarcoma

Isohexenylnaphthazarins are commonly found in the root periderm of several Boraginaceous plants and are known for their broad range of biological activities. The work described herein concerns the biological activity of compounds from the roots of Echium plantagineum L. and Echium gaditanum Boiss (Boraginaceae) collected from field sites in southern Spain and Australia. Bioactivity was assessed using etiolated wheat coleoptile bioassay and in vitro growth inhibitory activity in HeLa and IGROV-1 cells. The quantification of four isohexenylnaphthazarins (shikonin/alkannin, deoxyshikonin/deoxyalkannin, acetylshikonin/acetylalkannin and dimethylacrylshikonin/dimethylacrylalkannin) was performed by LC-MS/MS using juglone as internal standard. Correlation coefficient values for the activities and concentrations of these four analytes were in the linear range and were greater than 0.99. Acetylshikonin/acetylalkannin and dimethylacrylshikonin/dimethylacrylalkannin were present in the highest concentrations in extracts of both species. The results reveal that greatest overall inhibition was observed in both bioassays with E. gaditanum extracts. Strong correlations between time of collection, sampling location and bioactivity were identified.

Topics: Australia; Cell Line, Tumor; Cell Survival; Echium; Humans; Naphthoquinones; Plant Extracts; Plant Roots; Spain; Triticum

Cholangiocarcinoma (CCA), arising from varying locations within the biliary tree, is the second most common primary liver malignancy worldwide. Shikonin, an active compound extracted from the Chinese herb Zicao, holds anti-bacterial, anti-inflammatory, and anti-tumor activities. However, the effect of shikonin on human cholangiocarcinoma and detailed mechanisms of TRAIL enhancement remains to be elucidated. The purpose of the study was to investigate the protective functions of TRAIL enhancement for shikonin induced apoptosis in cholangiocarcinoma cells.. We use MTT assay, apoptosis assay, caspase activity assay, flow cytometry assay, real time PCR and Western blot to observe the effects of TRAIL on shikonin induced cholangiocarcinoma cells apoptosis and its mechanism.. Shikonin inhibited cell viability and induced apoptosis of CCA cells, effects enhanced by TRAIL treatment via activation of caspase-3, -8, -9. Furhermore, TRAIL enhanced anti-proliferation of shikonin and shikonin induced apoptosis through induction of ROS mediated JNK activation, while AKT activation had an effect on shikonin anti-proliferation activity, but not in the TRAIL enhanced counterparts. Finally, shikonin upregulated DR5 expression, an effect essential for TRAIL-enhanced activities of shikonin in RBE cells.. Our results revealed that shikonin could inhibit cells viability and induce apoptosis of CCA cells, effects enhanced by TRAIL treatment via ROS mediated JNK signalling pathways, involving up-regulation of DR5 expression. Our results provide further insight into the mechanism underlying the anti-tumor effects of shikonin by TRAIL enhanced in CCA and a new therapeutic strategy to CCA treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bile Duct Neoplasms; Bile Ducts; Cell Line; Cell Line, Tumor; Cholangiocarcinoma; Humans; MAP Kinase Kinase 4; Naphthoquinones; Reactive Oxygen Species; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

Shikonin and its derivatives extracted from Lithospermeae plants' red roots have current applications in food and pharmaceutical industries. Previous studies have cloned some genes related to shikonin biosynthesis. However, most genes related to shikonin biosynthesis remain unclear, because the lack of the genome/transcriptome of the Lithospermeae plants. Therefore, in order to provide a new understanding of shikonin biosynthesis, we obtained transcriptome data and unigenes expression profiles in three shikonin-producing Lithospermeae plants, i.e., Lithospermum erythrorhizon, Arnebia euchroma and Echium plantagineum. As a result, two unigenes (i.e., G10H and 12OPR) that are involved in "shikonin downstream biosynthesis" and "methyl jasmonate biosynthesis" were deemed to relate to shikonin biosynthesis in this study. Furthermore, we conducted a Lamiids phylogenetic model and identified orthologous unigenes under positive selection in above three Lithospermeae plants. The results indicated Boraginales was more relative to Solanales/Gentianales than to Lamiales.

Topics: Biological Evolution; Biosynthetic Pathways; Boraginaceae; Chromatography, High Pressure Liquid; Computational Biology; Gene Expression Profiling; Gene Expression Regulation, Plant; High-Throughput Nucleotide Sequencing; Lithospermum; Molecular Sequence Annotation; Naphthoquinones; Phylogeny; Selection, Genetic; Transcriptome

Hypoxia enhances the development of solid tumors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor of tumor regulation. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, and cell proliferation, as well as imparting resistance to cancer treatment. In this study, we assessed shikonin, which derives from the traditional medical herb Lithospermum erythrorhizon, for its anti-cancer effects in hypoxia-induced human colon cancer cell lines. Shikonin showed potent inhibitory activity against hypoxia-induced HIF-1α activation in various human cancer cell lines and efficient scavenging activity of hypoxia-induced reactive oxygen species in tumor cells. Further analysis revealed that shikonin inhibited HIF-1α protein synthesis without affecting the expression of HIF-1α mRNA or degrading HIF-1α protein. It was subsequently shown to attenuate the activation of downstream mTOR/p70S6K/4E-BP1/eIF4E kinase. Shikonin also dose-dependently caused the cell cycle arrest of activated HCT116 cells and inhibited the proliferation of HCT116 and SW620 cells. Moreover, it significantly inhibited tumor growth in a xenograft modal. These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; HCT116 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lithospermum; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Neoplasms; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases; Transplantation, Heterologous

This study aimed to examine the antiviral effects of shikonin ester ((R)-1-(5, 8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl3-(1H- indol-3-yl) propanoate (PMM-034) against influenza A (H1N1) virus. We investigated PMM-034 anti-H1N1 activity and its effect on caspase 3 gene expression during cellular apoptosis after influenza virus infection in vitro. Neuraminidase (NA) inhibition was assessed in comparison with oseltamivir in the influenza virus standard strains A/PR/8/34 to understand the viral mechanism. MDCK and A549 cells were used to investigate influenza viral infection and the structure-activity relationship between PMM-034 and NA was evaluated by pharmacophore-based docking modeling. The production of viral protein was tested by western blot. A/PR/8/34 induced cell inhibition but this was reduced by PMM-034 to 16μg/mL and this showed a selective index of 10mM. PMM-034 inhibited NA in a dose dependent manner, similar to oseltamivir inhibition. A sharp decrease in viral nucleocapsid protein mRNA was observed in infected cells after treatment with PMM-034. Apoptosis of infected A459 cells was inhibited by PMM-034 with decreased caspase 3 levels. ARG 118, ARG 152, ARG 371 and GLU 227 in the binding pocket of NA bound to PMM-034 in the docking model. Taken together, these results suggest PMM-034 shikonin ester blocked H1N1 infection and might be a potential anti-H1N1 drug.

Topics: A549 Cells; Animals; Antiviral Agents; Apoptosis; Cell Line; Cell Line, Tumor; Dogs; Enzyme Inhibitors; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Madin Darby Canine Kidney Cells; Naphthoquinones; Orthomyxoviridae Infections; Oseltamivir; Structure-Activity Relationship; Viral Proteins

We aimed to find candidate molecules possibly involved in the anti-inflammatory activity of shikonin (active compound of "Shikon") by analyzing its effects on gene expression of lipopolysaccharide (LPS)-treated THP-1 macrophages. Polysome-associated mRNAs (those expected to be under translation: translatome) from cells treated with LPS alone (LPS: 5 µg/mL), shikonin alone (S: 100 nM), or LPS plus shikonin (LPS&S) for 3 h were analyzed by DNA microarray followed by detection of enriched pathways/gene ontologies using the tools of the STRING database. Candidate genes in enriched pathways in the comparison of LPS&S cells vs. LPS cells were analyzed by reverse-transcription quantitative real-time PCR (RT-qPCR; 1, 2, and 3 h). DNA microarray showed shikonin significantly influences gene expression. Gene expression changes between LPS&S cells and LPS cells were compared to detect relevant proteins and/or mRNAs underlying its anti-inflammatory effects: shikonin downregulated pathways which were upregulated in LPS cells, for example, 'innate immune response'. Within changed pathways, three genes were selected for RT-qPCR analyses as key candidates influencing inflammatory responses: CYBA (component of the superoxide-generating Nox2 enzyme), GSK3B (controller of cell responses after toll-like receptor stimulation), and EIF4E (a key factor of the eukaryotic translation initiation factor 4F complex that regulates abundance of other proteins involved in immune functions). All three mRNAs were decreased at 2 h, and CYBA continued low at 3 h relative to LPS cells. Given that shikonin decreased the expression of CYBA gene of Nox2, in addition to the direct inhibition of the Nox2 activity that we have previously shown, it is suggested that one of its anti-inflammatory mechanisms could be attenuation of oxidative stress.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Differentiation; Gene Expression; Humans; Inflammation; Lipopolysaccharides; Macrophages; Naphthoquinones

Acute promyelocytic leukemia (APL) is a special subtype of acute myeloid leukemia that responds to treatment with all‑trans retinoic acid and arsenic trioxide. However, severe side effects and drug resistance limit the effectiveness of these treatments. Hence, new drugs for APL are required urgently. Shikonin, an active naphthoquinone derived from the Chinese medical herb Zi Cao exerts antitumor activity in several cancers. In the present study, the effects of shikonin on proliferation and apoptosis in NB4 cells, as well as related mechanisms were assessed. Treatment of NB4 cells with shikonin inhibited proliferation in a concentration‑ and time‑dependent manner. The cell cycle was arrested in the G1 phase. NB4 cells treated with shikonin exhibited more apoptosis and higher levels of cleaved caspase‑3 and poly ADP‑ribose polymerase than control cells. Western blotting results demonstrated that the expression of p‑p38 mitogen‑activated protein kinase (p‑p38MAPK) and p‑c‑Jun N‑terminal kinase (p‑JNK) was increased significantly by shikonin treatment, while the expression of p‑ERK and c‑Myc was decreased. In summary, these findings indicated that shikonin inhibited cell proliferation and induced apoptosis partly through modulation of the MAPKs and downregulation of c‑Myc.

Topics: Apoptosis; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Nucleus Shape; Cell Proliferation; Down-Regulation; Humans; Leukemia, Promyelocytic, Acute; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Naphthoquinones; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-myc

Pyruvate kinase M2 (PKM2) regulates glycolysis and oxidative phosphorylation; however, the role of PKM2 in ovarian cancer remains largely unknown. We investigated whether ovarian cancer metabolism could provide insight into the development of therapeutic strategies. We performed immunohistochemical staining for PKM2 on a tissue microarray for multivariate analysis. It revealed that patients exhibiting higher PKM2 expression were significantly associated with malignancy groups (p < 0.001) and pathogenesis models (p < 0.001), had poor progression-free survival rates (p = 0.01) as compared with patients exhibiting lower PKM2 levels, and yielded a hazard ratio of death of 2.02 (95% confidence interval: 0.70-5.85). In cell lines, PKM2 inhibitor significantly inhibited the glycolytic rate according to cellular glucose consumption (p < 0.001). We also utilized Seahorse assays to assess metabolism-related cell-specific factors and the impact of PKM2 inhibitors. Energy shifts as per Seahorse analysis showed attenuation of the extracellular acidification rate (p < 0.05) and no significant difference in oxygen-consumption rate in SKOV3 cells. Treatment with PKM2 inhibitor suppressed ovarian cancer growth and cell migration in vitro and inhibited tumor growth without significant toxicity in a xenograft study. PKM2 inhibition disturbed Warburg effects and inhibited ovarian cancer cell growth. Targeting PKM2 may constitute a promising therapy for patients with ovarian cancer, and clinical trials involving shikonin are warranted.

Topics: Animals; Biomarkers; Cell Line, Tumor; Cell Survival; Disease-Free Survival; Enzyme Inhibitors; Female; Glucose; Humans; Immunohistochemistry; Lactic Acid; Mice, SCID; Naphthoquinones; Ovarian Neoplasms; Positron-Emission Tomography; Prognosis; Pyruvate Kinase; Tissue Array Analysis; Xenograft Model Antitumor Assays

Shikonin, isolated from the roots of herbal plant Lithospermum erythrorhizon, is a naphthoquinone. It has been reported to exert beneficial anti-inflammatory effects and anti-oxidant properties in various diseases. Isoproterenol (ISO) has been widely used to establish cardiac injury in vivo and in vitro. However, shikonin function in ISO-induced cardiac injury remains uncertain. In our study, we attempted to investigate the efficiency and possible molecular mechanism of shikonin in cardiac injury treatment induced by ISO. In vivo, C57BL6 mice were subcutaneously injected with 5mg/kg ISO to induce heart failure. And mice were given a gavage of shikonin (2 or 4mg/kg/d, for four weeks). Cardiac function, fibrosis indices, inflammation response, apoptosis and endoplasmic reticulum (ER) stress were calculated. Pathological alterations, fibrosis-, inflammation-, apoptosis- and ER stress-related molecules were examined. In ISO-induced cardiac injury, shikonin significantly ameliorated heart function, decreased myocardial fibrosis, suppressed inflammation, attenuated apoptosis and ER stress through impeding collagen accumulation, Toll like receptor 4/nuclear transcription factor κB (TLR4/NF-κB), Caspase-3 and glucose-regulated protein 78 (GRP78) signaling pathways activity, relieving heart failure in vivo. Also, in vitro, shikonin attenuated ISO-induced cardiac muscle cells by reducing fibrosis, inflammation, apoptosis and ER stress. Our findings indicated that shikonin treatment attenuated ISO-induced heart injury, providing an effective therapeutic strategy for heart failure treatment for future.

Topics: Animals; Apoptosis; Cardiomyopathies; Caspase 3; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Fibrosis; Gene Expression Regulation; Heart; Heart Failure; HSP70 Heat-Shock Proteins; Inflammation; Isoproterenol; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Myocardium; Myocytes, Cardiac; Naphthoquinones; NF-kappa B

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 (RIP1) and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species (ROS), but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin (2-10 μmol/L) dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose-dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 (100 μmol/L) or the specific RIP3 inhibitor GSK-872 (5 μmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 μmol/L), which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.

Topics: Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Glioma; Humans; Mitochondria; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Oxidative Stress; Protein Serine-Threonine Kinases; Rats; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Signal Transduction; Time Factors

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants and children below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activities derived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviral activity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 on RD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cells were determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions were determined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibited significant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 μg/mL. The VP1 mRNA and protein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1β, IL-6, IL-8, and TNF-α significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression was also significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.

Topics: Antiviral Agents; Blotting, Western; Cell Line, Tumor; Cytokines; Dose-Response Relationship, Drug; Enterovirus A, Human; Humans; Naphthoquinones; Real-Time Polymerase Chain Reaction; Rhabdomyosarcoma; Toxicity Tests; Viral Plaque Assay; Virus Replication

Fibroblast is believed to be the primary effector in idiopathic pulmonary fibrosis (IPF), a progressive lung disorder characterized by aberrant tissue remodeling and the formation of fibroblastic foci. Due to the complicated etiology and mechanism, there are few effective drugs for this fatal disease. Shikonin (SHI), which is the major ingredient isolated from the plant Lithospermum Erythrorhizon, has long been used as traditional medicine for many diseases including inflammation and cancer. The roles of SHI in attenuating skin scar and renal fibrosis by reducing TGFβ1-stimulated fibroblast activation are also reported. But whether SHI works on IPF which exhibits both inflammatory and carcinoma-like features remains unknown. In this study, using isolated pulmonary fibroblasts, we demonstrated that SHI inhibited the proliferation, migration of fibroblasts, enhanced cell apoptosis and led to cell cycle arrest at G1 and G2/M phase. Moreover, SHI reduced the production of α-SMA, fibronectin, collagen I and III in response to TGF-β induction in pulmonary fibroblasts, and all of these gene production is the key component of extracellular matrix for tissue remodeling for IPF. The phosphorylation of Akt was down-regulated, p53 increased, the mRNA levels of p21 and p27 enhanced after SHI treatments. The phosphorylation of both p38 MAPK and Akt stimulated by TGF-β was reduced after SHI treatments. Collectively, these data indicate that SHI has a strong cytotoxicity in pulmonary fibroblast via inhibiting Akt activation signaling pathway, and attenuates TGF-β induced extracellular matrix genes production in pulmonary fibroblasts via modulating the activities of p38 MAPK and Akt. SHI might serve as a therapeutically candidate for IPF patients.

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Cell Survival; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation; Lung; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta1

Shikonin and its derivatives are the main active components in the medicinal plant Arnebia euchroma and possess extensive pharmaceutical properties. In this study, we developed an optimized yeast system to obtain high-level production of 3-geranyl-4-hydroxybenzoate acid (GBA), an important intermediate involved in shikonin biosynthesis pathway. For host selection, recombinant expression of p-hydroxybenzoate:geranyltransferase (PGT) derived from A. euchroma was performed in Saccharomyces cerevisiae WAT11U strain and high yield monoterpene strain. In shake flask culture with 1 mM p-hydroxybenzoate acid (PHBA), they could yield GBA at 0.1567 and 20.8624 mg L-1, respectively. Additionally, AePGT6 showed higher enzymatic activity than its homologs. Moreover, by combining improvement in the homologous mevalonate pathway with reconstruction in the heterologous shikimic pathway, a de novo GBA synthesis pathway was constructed in StHP6tHC with co-overexpressed SctHMG1, optimized EcUbiC and AePGT6. A high titer of 179.29 mg L-1 GBA was achieved in StHP6tHC under shake flask fermentation with 1 mM PHBA. These results suggest that yeast could be engineered systematically to enable an efficient monoterpene-quinone or naphthoquinone production.

Topics: Biosynthetic Pathways; Biotransformation; Chromatography, High Pressure Liquid; Enzyme Activation; Fermentation; Gene Expression; Geranyltranstransferase; Metabolic Engineering; Naphthoquinones; Parabens; Saccharomyces cerevisiae; Yeasts

Shikonin, a compound extracted from Zicao, has been demonstrated to hold anti-bacterial, anti-inflammatory, and anti-tumor activities in various diseases and it has been shown to protect human organs from injuries. However, the effect of shikonin on the recovery of spinal cord injury (SCI) remains unknown. This study was designed to estimate the potential therapeutic effect and underlying mechanism of shikonin on SCI in vivo.. In the study, we used HE staining, ELISA assay, transfection assay, TUNEL assay, real time PCR and Western blot to detect the effects of shikonin on spinal cord injury in rats.. we showed that shikonin could promote the recovery of motor function and tissue repair after SCI treatment in rats SCI model. Moreover, we demonstrated that shikonin inhibited the spinal cord edema in SCI model of rats. According to further investigation, shikonin induced the reduction of inflammatory response through decreasing the expression levels of HMGB1, TLR4 and NF-κB after SCI injury. In addition, we also found that shikonin could suppress the apoptosis and expression of caspase-3 protein in SCI model of rats.. Our results demonstrated that shikonin induced the recovery of tissue repair and motor function via inactivation of HMGB1/TLR4/NF-κB signaling pathway in SCI model of rats. Meanwhile, shikonin regulated the inflammation response in SCI by suppressing the HMGB1/TLR4/NF-κB signaling pathway. The described mechanism sheds novel light on molecular signaling pathway in spinal cord injury and secondary injury including inflammatory response.

Topics: Animals; Anti-Inflammatory Agents; HMGB1 Protein; Male; Naphthoquinones; NF-kappa B; Rats; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; Spinal Cord; Spinal Cord Injuries; Toll-Like Receptor 4

Shikonin is a naphthoquinone pigment extracted from roots of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), and possesses various pharmaceutical activities, such as anti-inflammation and anti-cancer effects. In addition, shikonin as a natural red colorant for food garnishment and cosmetics ingredient is widely used in the world. However, the inhibition risk of shikonin on cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the potential inhibition of shikonin against CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver microsomes through cocktail approach in vitro. The results demonstrated that shikonin exhibited no time-dependent inhibition of CYP activities. In human liver microsomes, shikonin was not only a mixed inhibitor of CYP1A2, CYP2B6, CYP2C9, CYP2D6 and CYP3A4, but also a competitive inhibitor of CYP2E1, with K

Topics: Animals; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Interactions; Food-Drug Interactions; Humans; Hydroxylation; Inhibitory Concentration 50; Male; Microsomes, Liver; Naphthoquinones; Rats; Rats, Sprague-Dawley

An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments.. Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry.. We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results.. In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

Topics: Alternative Splicing; Animals; Antagomirs; Cattle; Cell Proliferation; Endothelium, Vascular; Fibroblasts; Glycolysis; Heterogeneous-Nuclear Ribonucleoproteins; Histone Deacetylase Inhibitors; Humans; Hypertension, Pulmonary; Interleukin-1beta; Macrophages; Mice; Mice, Inbred C57BL; MicroRNAs; Naphthoquinones; Polypyrimidine Tract-Binding Protein; Protein Isoforms; Pyruvate Kinase; RNA Interference

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, is considered to have antitumor effects. Gallbladder cancer (GBC) is a prevalent biliary tract malignancy with few curative therapeutic stragegies and poor prognosis. In the present study, we detected the effects of shikonin on GBC cells as well as the underlying molecular mechanisms. The results demonstrated that GBC cell proliferation was inhibited by shikonin as determined by MTT and colony formation assays. Flow cytometry results demonstrated that shikonin treatment enhanced apoptosis and promoted G0/G1 phase arrest in the GBC cells. Western blot assay showed that shikonin induced mitochondrial-dependent apoptosis via the JNK signaling pathway. Moreover, shikonin suppressed tumor growth in mice bearing GBC-derived xenografts in a dose‑dependent manner without side-effects. These results revealed that shikonin exhibits anticancer effects on GBC cells by inducing apoptosis and regulating the cell cycle. Taken together, shikonin may be a novel and safe chemotherapeutic agent for the treatment of GBC.

Topics: Animals; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; G1 Phase Cell Cycle Checkpoints; Gallbladder Neoplasms; Humans; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mice; Naphthoquinones; Xenograft Model Antitumor Assays

The signal transducer and activator of transcription 3 is a constitutively activated oncogenic protein in various human tumors and represents a valid target for anticancer drug design. In this study, we have achieved a new type of STAT3 inhibitors based on structural modifications on shikonin scaffold, guided by computational modelling. By tests, PMMB-187 exhibited a more outstanding profile than shikonin on a small panel of human breast cancer cells, especially for the MDA-MB-231 cells. For the cellular mechanisms research, PMMB-187 was found to induce cell apoptosis in MDA-MB-231 cells, associated with the reduction of mitochondrial membrane potential, production of ROS and alteration of the levels of apoptosis-related proteins. Furthermore, PMMB-187 inhibited constitutive/inducible STAT3 activation, transcriptional activity, nuclear translocation and downstream target genes expression in STAT3-dependent breast cancer cells MDA-MB-231. Besides, no obvious inhibitory effect on activation of STAT1 and STAT5 was observed with PMMB-187 treatment. Most notably, the in vivo studies further revealed that PMMB-187 could dramatically suppress the MDA-MB-231 cells xenografted tumor growth. The in vitro and in vivo results collectively suggest that PMMB-187 may serve as a promising lead compound for the further development of potential therapeutic anti-neoplastic agents.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Membrane Potential, Mitochondrial; Models, Molecular; Molecular Structure; Naphthoquinones; STAT3 Transcription Factor; Thiadiazoles

Shikonin is a naphthoquinone secondary metabolite with important medicinal value and is found in Lithospermum erythrorhizon. Considering the limited knowledge on the membrane transport mechanism of shikonin, this study investigated such molecular mechanism.. We successfully isolated an ATP-binding cassette protein gene, LeMDR, from L. erythrorhizon. LeMDR is predominantly expressed in L. erythrorhizon roots, where shikonin accumulated. Functional analysis of LeMDR by using the yeast cell expression system revealed that LeMDR is possibly involved in the shikonin efflux transport. The accumulation of shikonin is lower in yeast cells transformed with LeMDR-overexpressing vector than that with empty vector. The transgenic hairy roots of L. erythrorhizon overexpressing LeMDR (MDRO) significantly enhanced shikonin production, whereas the RNA interference of LeMDR (MDRi) displayed a reverse trend. Moreover, the mRNA expression level of LeMDR was up-regulated by treatment with shikonin and shikonin-positive regulators, methyl jasmonate and indole-3-acetic acid. There might be a relationship of mutual regulation between the expression level of LeMDR and shikonin biosynthesis.. Our findings demonstrated the important role of LeMDR in transmembrane transport and biosynthesis of shikonin.

Topics: ATP-Binding Cassette Transporters; Biological Transport; Blotting, Southern; Cloning, Molecular; Gene Expression Regulation, Plant; Genes, Plant; Lithospermum; Naphthoquinones; Plant Roots; Plants, Genetically Modified; Sequence Analysis, DNA

Non-small cell lung cancer (NSCLC) is the dominant type of lung cancer. Molecular targeting has highly improved the treatment efficacy of lung cancer, but new challenges have emerged, such as gefitinib-resistance and cancer recurrence. Therefore, new chemotherapeutic agents and treatment strategies are urgently needed. Shikonin is the main active component of a Chinese medicinal plant 'Zi Cao', which has been shown to exhibit powerful anti-cancer activity in certain types of cancer; however, its activity in gefitinib-resistant lung cancer has never been addressed. In this study, we used a high-throughput screening assay for epidermal growth factor receptor (EGFR) inhibitors and discovered that Shikonin is a potent inhibitor of EGFR. The cytotoxicity of Shikonin and its anti-cancer mechanism in NSCLC was deeply explored. Shikonin exhibited selective cytotoxicity among two NSCLC cell lines (H1975 and H1650) and one normal lung fibroblast cell line (CCD-19LU). Shikonin significantly increased the activity of caspases and poly (ADP-ribosyl) polymerase (PARP), which are indicators of apoptosis, and the intensity of ROS by greater than 10-fold. NAC, an inhibitor of ROS, completely blocked apoptosis, caspase and PARP activation induced by Shikonin. Shikonin remarkably suppressed the phosphorylation of EGFR and led to EGFR degradation. The enhancement of ROS generation in H1650 and H1975 gefitinib-resistant NSCLC cells leads to impairment of growth and induction of apoptosis, whereas modulation of EGFR degradation and its downstream signalling pathways by Shikonin contributes to its anti-tumour properties in H1975 gefitinib-resistant NSCLC cells (with T790M and L858R activating mutations). Shikonin-induced cell apoptosis is closely associated with ROS elevation in the cells. These findings indicate that Shikonin can be an effective small molecule treating gefitinib-resistant NSCLC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; High-Throughput Screening Assays; Humans; Lung Neoplasms; Mutation; Naphthoquinones; Neoplasm Recurrence, Local; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; Signal Transduction; Thioredoxin-Disulfide Reductase

Shikonin, a major effective component in the Chinese herbal medicine Lithospermum erythrorhizon Sieb., exhibits an anti-inflammatory property towards rheumatoid arthritis (RA), but the potential mechanism is unclear. Our aim was to investigate the mechanism of shikonin on the lipopolysaccharide (LPS)-induced fibroblast-like synoviocyte (LiFLS) inflammation model. Fibroblast-like synoviocytes (FLSs) were treated with 200 μg/ml of LPS for 24 h to establish the RA-like model, LiFLS. FLSs were pretreated with shikonin (0.1-1 μM) for 30 min in the treatment groups. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays were used to detect mRNA and protein levels of interleukin (IL)-10 and tumor necrosis factor (TNF)-α. Signal proteins involved in IL-10 production were analyzed by Western blotting. Shikonin significantly reversed the inhibitory effects of LPS on IL-10 expression in FLSs by inactivating the PKC-NF-κB pathway. In addition, shikonin inhibited LPS-induced TNF-α expression in FLSs, and this effect was markedly diminished by IL-10-neutralizing antibody. The IL-10-mediated suppression of TNF-α transcription was demonstrated by no response to the protein synthesis inhibitor cyclohexamide and no mRNA decay. Shikonin inhibits LPS-induced TNF-α production in FLSs through suppressing the PKC-NF-κB-dependent decrease in IL-10, and this study also highlights the potential application of shikonin in the treatment of RA.

Topics: Arthritis, Rheumatoid; Drugs, Chinese Herbal; Humans; Interleukin-10; Naphthoquinones; NF-kappa B; Tumor Necrosis Factor-alpha

SIRT2 is involved in the development of a variety of cancers. Shikonin is a natural compound that is known to have antitumor effects. This study aims to assess the effects of shikonin on the development and metastatic progression of colorectal cancer (CRC) through regulation of SIRT2 expression and whether this effect is related to the phosphorylation of extracellular signal-regulated kinases (ERKs). The results demonstrated that SIRT2 is downregulated in CRC biopsy samples (n=31) compared with the adjacent non-cancerous tissues (ANCT, n=26). Furthermore, CRC metastases were positive for SIRT2 despite a lack of expression in the primary tumor. In addition, data from an in vitro assay revealed that overexpression of SIRT2 inhibited the proliferation and metastatic progression of SW480 cells while blocking of SIRT2 expression induced the proliferation and metastatic progression of HT29 cells. Shikonin inhibited the viability, migration and invasion of SW480 cells and it also inhibited the tumor growth in the nude mice model; while AGK2 (a specific inhibitor of SIRT2) reversed these effects. Epidermal growth factor (EGF, an activator of ERK) and ERK-overexpression inhibited the effects of shikonin on SIRT2 expression, proliferation and metastasis in SW480 cells. However, this proliferative effect of EGF was reversed by SIRT2 overexpression. In conclusion, these results suggest that SIRT2 is a new therapeutic target for the treatment of CRC. The antitumor effects of shikonin on CRC seem to be mediated by SIRT2 upregulation via phospho-ERK inhibition.

Topics: Animals; Antineoplastic Agents; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Colorectal Neoplasms; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Middle Aged; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Sirtuin 2

Shikonin has been reported to induce glioma cell death via necroptosis, a type of programmed necrosis primarily mediated by RIP1 and RIP3. Although RIP1 and RIP3 are found to regulate some features of necrosis such as energy depletion and cellular membrane disruption, it remains unclear whether RIP1 and RIP3 could modulate DNA double strand breaks (DSBs), which is a crucial event leading to chromatinolysis. In this study, we used glioma cell lines and mice model of xenograft glioma to investigate the roles of RIP1 and RIP3 in shikonin-induced DNA DSBs. We found that shikonin induced upregulation of RIP1 and RIP3, necrosome formation and DNA DSBs in vitro and in vivo. In vitro investigation showed that the DNA DSBs and the reduction of cellular viabilities induced by shikonin were both prevented when RIP1 or RIP3 was pharmacologically inhibited by specific inhibitor or genetically knocked down with siRNA. Then, we proved that suppression of intracellular ROS with antioxidant NAC inhibited DNA DSBs caused by either hydrogen peroxide or shikonin, suggesting that ROS played a crucial role in regulation of DNA DSBs of glioma cells induced by shikonin. Further, we found that RIP1 and RIP3 regulated shikonin-induced overproduction of ROS via causing excessive generation of mitochondrial superoxide and depletion of GSH, indicating that ROS was the downstream signal of RIP1 and RIP3. Taken together, we demonstrated that RIP1 and RIP3 contributed to shikonin-induced DNA DSBs in glioma cells via increase of intracellular ROS levels.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; DNA Breaks, Double-Stranded; Glioma; Heterografts; Mice; Naphthoquinones; Nuclear Pore Complex Proteins; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Up-Regulation

Topics: Antineoplastic Agents; Apoptosis; Binding Sites; Cell Line; Cell Proliferation; Chalcone; Drug Design; G2 Phase Cell Cycle Checkpoints; Humans; M Phase Cell Cycle Checkpoints; MCF-7 Cells; Membrane Potential, Mitochondrial; Microscopy, Confocal; Molecular Docking Simulation; Naphthalenes; Naphthoquinones; Protein Structure, Tertiary; Structure-Activity Relationship; Tubulin; Tubulin Modulators

Chronic kidney diseases generally lead to renal fibrosis. Despite great progress having been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear, and so far no effective therapeutic antifibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes was upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in transforming growth factor-β1 (TGF-β1)-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGF-β1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkably induce myofibroblast activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose attenuate UUO-induced mouse renal fibrosis and TGF-β1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2, a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and support treatment with aerobic glycolysis inhibitors as a potential antifibrotic strategy.

Topics: Animals; Carrier Proteins; Cell Line; Deoxyglucose; Disease Models, Animal; Fibrosis; Glycolysis; Kidney; Male; Membrane Proteins; Mice; Myofibroblasts; Naphthoquinones; Phosphorylation; Pyruvate Kinase; Rats; Renal Insufficiency, Chronic; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Time Factors; Transforming Growth Factor beta1; Ureteral Obstruction

Metabolic profiling can be successfully implemented to analyse a living system's response to environmental conditions by providing critical information on an organism's physiological state at a particular point in time and allowing for both quantitative and qualitative assessment of a specific subset(s) of key metabolites. Shikonins are highly reactive chemicals that affect various cell signalling pathways and possess antifungal, antibacterial and allelopathic activity. Based on previous bioassay results, bioactive shikonins, are likely to play important roles in the regulation of rhizosphere interactions with neighbouring plants, microbes and herbivores. An effective platform allowing for rapid identification and accurate profiling of numerous structurally similar, difficult-to-separate bioactive isohexenylnaphthazarins (shikonins) was developed using UHPLC Q-TOF MS. Root periderm tissues of the invasive Australian weeds

Topics: Australia; Chromatography, High Pressure Liquid; Echium; Metabolome; Metabolomics; Molecular Structure; Naphthoquinones; Plant Extracts; Plant Roots; Plant Weeds; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Shikonin and its enantiomer alkannin, which are natural products, have been extensively studied in vitro and in vivo for, among others, their antitumor activity. The investigation of the molecular pathways involved in their action is of interest, since they are not yet clearly defined. Metabolic profiling in cells can provide a picture of a cell's phenotype upon intervention, assisting in the elucidation of the mechanism of action. In this study, the cytotoxic effect of shikonin on a human hepatocarcinoma cell line was studied. Huh7 cells were treated with shikonin at 5 μM, and it was found that shikonin markedly inhibited cell growth. Metabolic profiling indicated alterations in the metabolic content of the cells and the culture media upon treatment, detecting the metabolic response of the cells. This study demonstrates the potential of metabolomics to improve knowledge on the mechanisms involved in shikonin's antitumor action.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, Liquid; Humans; Liver Neoplasms; Metabolome; Metabolomics; Naphthoquinones; Signal Transduction; Tandem Mass Spectrometry

Shikonin is a kind of naphthoquinone compound found mainly in Lithospermum erythrorhizon Sieb,et Zucc. Previous studies have shown that Shikonin has anti-tumor, anti-inflammatory and extensive pharmacological effects. According to new studies, Shikonin could also modulate the immune system function, but the effect to NK (nature killer) cells is yet unknown.. To investigate the effect and mechanism of Shikonin on NK cells proliferation and cytotoxicity to colon cancer cell line (Caco-2).. The proliferation and cytotoxicity of NK cells cultured with Shikonin were detected with CCK-8 assay. The expressions of perforin, GranB and IFN-γ were examined with FCM. The content of TNF-alpha was disclosed with ELISA kit. p-ERK1/2 and p-Akt expression of NK cells were detected with western blot.. With CCK-8 assay, it is found that Shikonin could significantly enhance NK cells proliferation and cytotoxicity to colon cancer cells. With FCM assay, it is found that Shikonin could improve the expression of perforin and GranB in a dose-dependent manner. Shikonin had no effect on TNF-alpha and IFN-γ expression. In mechanism, the study shows that Shikonin could enhance the expression of p-ERK1/2 and p-Akt.. Shikonin enhances NK cells proliferation and cytotoxicity via the improvement of perforin, GranB, p-ERK1/2 and p-Akt expression.

Topics: Caco-2 Cells; Cell Proliferation; Colonic Neoplasms; Humans; Immunity, Cellular; Killer Cells, Natural; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; Proto-Oncogene Proteins c-akt

The p-hydroxybenzoate geranyltransferases(PGT) play an important role in the biosynthesis pathways of shikonin derivatives. Six PGTs were obtained from transcriptome datebase of Arnebia euchroma by using bioinformatics methods and the proteins＇physiochemical properties they encoded were predicted. The result of protein domain prediction showed all of the six protein sequences contained the conserved domain of Ubia prenyltransferase family and possessed the motif NDxxDxxxD for prenyldiphosphate binding and a GX(K/Y)STAL sequence for putative aromatic ring binding. The phylogenetic tree showed that PGT and p-hydroxybenzoate polyprenyltransferase(PPT) belonged to two different clades. The results of gene expression analyses showed that the expression levels in the red shikonin-proficient line and the overground part of A. euchroma that could produce shikonin derivatives was much higher than the white shikonin-deficient line and the underground part, which suggested a positive correlation between the expression levels of PGT genes and shikonin production. This study aims to lay a foudation for further understanding of the function and enzymatic properties of PGT and provide a basis for the biosynthesis pathways and metabolic regulation of shikonin derivatives.

Topics: Boraginaceae; Computational Biology; Geranyltranstransferase; Naphthoquinones; Parabens; Phylogeny; Transcriptome

Resistance to cell death and reprogramming of metabolism are important in neoplastic cells. Increased resistance to apoptosis and recurrence of tumors are the major roadblocks to effective treatment of triple negative breast cancer. It has been thought that execution of necroptosis involves ROS generation and mitochondrial dysfunction in malignant cells. In this study, the effect of shikonin, an active substance from the dried root of Lithospermum erythrorhizon, on the induction of necroptosis or apoptosis, via RIP1K-RIP3K expressions has been examined in the triple negative breast cancer cell line. The expression levels of RIP1K and RIP3K, caspase-3 and caspase-8 activities, the levels of ROS, and mitochondrial membrane potential have been studied in the shikonin-treated MDA-MB-468 cell line. An increase in the ROS levels and a reduction in mitochondrial membrane potential have been observed in the shikonin-treated cells. Cell death has mainly occurred through necroptosis with a significant increase in the RIP1K and RIP3K expressions, and characteristic morphological changes have been observed. In the presence of Nec-1, caspase-3 mediating apoptosis has occurred in the shikonin-treated cells. The current findings have revealed that shikonin provoked mitochondrial ROS production in the triple negative breast cancer cell line, which works as a double-edged executioner's ax in the execution of necroptosis or apoptosis. The main route of cell death induced by shikonin is RIP1K-RIP3K-mediated necroptosis, but in the presence of Nec-1, apoptosis has prevailed. The present results shed a new light on the possible treatment of drug-resistant triple negative breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 8; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Shape; Cell Survival; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Naphthoquinones; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Triple Negative Breast Neoplasms; Up-Regulation

Cisplatin-based therapy is one of the most important chemotherapy treatments for cancers. However, its efficacy is greatly limited by drug resistance and undesirable side effects. Therefore, it is of great importance to develop effective chemosensitization agents to cisplatin. In the present study, we demonstrated the strategy to use shikonin, a natural product from the root of Lithospermum erythrorhizon, as a synergistic agent of cisplatin and elucidated their action mechanisms. The combination of shikonin and cisplatin exhibited synergistic anticancer efficacy and achieved greater selectivity between cancer cells and normal cells. By inducing intracellular oxidative stress, shikonin potentiated cisplatin-induced DNA damage, followed by increased activation of mitochondrial pathway. In addition, inhibition of ROS reversed the apoptosis induced by shikonin and cisplatin, and recovered the depletion of mitochondrial membrane potential, which revealed the vital role of ROS in the synergism. Moreover, HCT116 xenograft tumor growth in nude mice was more effectively inhibited by combined treatment with shikonin and cisplatin. Our findings suggest that the strategy to apply shikonin as a synergistic agent to cisplatin could be a highly efficient way to achieve anticancer synergism by inducing intracellular oxidative stress. Shikonin may be a promising candidate as a chemosensitizer to cisplatin-based therapy for cancer treatments.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cisplatin; Colonic Neoplasms; Drug Synergism; Drugs, Chinese Herbal; Female; HT29 Cells; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Reactive Oxygen Species; Treatment Outcome

The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.

Topics: Cloning, Molecular; Computational Biology; DNA, Complementary; Ethylenes; Gene Expression Regulation, Plant; Lithospermum; Lyases; Naphthoquinones; Plant Proteins; Plant Roots; Plants, Genetically Modified; Signal Transduction

DMAKO-05((S)-1-((5E,8E)-5,8-bis(hydroxyimino)-1,4-dimethoxy-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-methylbutanoate) is a novel oxime derivative of shikonin, the major component extracted from Chinese herb Lithospermun erythrorhizon. Here, we report that DMAKO-05 had an antitumor activity against mouse melanoma cell line B16F0. Our studies indicated that DMAKO-05 not only inhibited B16F0 proliferation and migration but also led to cell cycle arrest at G1 phase and cell apoptosis, in which DMAKO-05 triggered mitochondrial-mediated apoptosis signal including caspase-9/3 and PARP. In response to DMAKO-05 treatment, the Akt-mediated survival signals were remarkably attenuated in B16F0 cells. Collectively, DMAKO-05 has a strong cytotoxicity in B16F0 cells via inhibiting Akt activation, inducing G1 arrest, and promoting B16F0 cell apoptosis. DMAKO-05 might serve as a potential candidate lead compound for melanoma.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Melanoma; Naphthoquinones; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

β-hydroxyisovalerylshikonin (β-HIVS), which is a natural naphthoquinone compound, is one of the main chemicals isolated from a therapeutic plant, Lithospermum erythrorhizon. In the present study, we demonstrated that β-HIVS inhibited the adipogenesis of 3T3-L1 cells through AMP-activated protein kinase (AMPK)-mediated modulation of sterol regulatory element binding protein (SREBP)‑1c. The anti-adipogenic effect of β-HIVS was accompanied by the increased phosphorylation of AMPK and precursor SREBP‑1c. In β-HIVS-treated 3T3-L1 cells, AMPK was activated and phosphorylated precursor SREBP‑1c, preventing the cleavage of precursor SREBP‑1c to mature SREBP‑1c. Expression of the fat-forming enzymes, acetyl-CoA carboxylase (ACC)1, fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD)1, which are transcribed by mature SREBP‑1c, were downregulated, resulting in reduced intracellular fat accumulation. The anti-adipogenic effect of β-HIVS was significantly attenuated by AMPK knockdown. Knockdown of AMPK using siRNA decreased the phosphorylation of precursor SREBP‑1c and increased the levels of mature SREBP. The levels of the fat-forming enzymes, ACC1, FAS and SCD1, as well as intracellular fat accumulation were also significantly increased by AMPK knockdown. These results suggest that β-HIVS activated AMPK, which was followed by the downregulation of mature SREBP‑1c and fat-forming enzymes, leading to the inhibition of adipogenesis.

Topics: 3T3-L1 Cells; Acetyl-CoA Carboxylase; Aldehyde Oxidoreductases; AMP-Activated Protein Kinases; Animals; Cell Survival; Mice; Naphthoquinones; Reverse Transcriptase Polymerase Chain Reaction; Stearoyl-CoA Desaturase; Sterol Regulatory Element Binding Protein 1

The aim of the present study was to investigate the effects of shikonin (SHI) on the induction of apoptosis in human TT medullary thyroid carcinoma cells, and to explore the role of mitochondrial signaling in this process. MTT, Annexin V‑phycoerythrin/7‑aminoactinomycin D staining, electron microscopy and JC‑1 probe staining were performed to analyze mitochondrial membrane potential, and western blot analysis was used to examine the activation of the mitochondrial signaling pathway, and the changes in mitochondrial apoptosis pathway‑associated protein expression. Following culture for 24‑72 h, treatment with various concentrations of SHI inhibited the proliferation of TT cells, in a dose‑ and time‑dependent manner. Transmission electron microscopy demonstrated the presence of typical apoptotic structures, as well as mitochondrial structural changes. The expression levels of apoptosis‑associated proteins caspase‑9, caspase‑3 and poly adenosine triphosphate ribose polymerase increased in a dose‑dependent manner following treatment with SHI. In addition, the mitochondrial membrane potential of the experimental group was significantly decreased, and the mitochondrial apoptosis pathway‑associated proteins were altered. A possible mechanism underlying SHI‑induced apoptosis through the mitochondrial signaling pathway is the regulation of B cell lymphoma 2 (Bcl‑2)/Bcl‑2‑associated protein X expression levels, resulting in the decrease in mitochondrial membrane potential and the activation of the caspase‑9/caspase‑3 enzyme‑associated reactions.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; G1 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Signal Transduction

Shikonin, which is a major ingredient of the traditional Chinese herb Lithospermum erythrorhizon, possesses various biological functions, including antimicrobial, anti-inflammatory, and antitumor activities. The present study aimed to determine the molecular mechanisms underlying the effects of shikonin on HaCaT cell apoptosis. Treatment with shikonin significantly inhibited the viability of HaCaT cells in a dose‑ and time‑dependent manner, and promoted cell cycle arrest at G0/G1 phase and apoptosis. In addition, shikonin treatment reduced the mitochondrial membrane potential and induced reactive oxygen species generation. The results of a western blot analysis demonstrated that shikonin significantly activated caspase 3 expression, downregulated B‑cell lymphoma 2 (Bcl‑2) expression, and upregulated Bcl‑2‑associated X protein and Bcl‑2 homologous antagonist killer expression in a dose‑dependent manner in HaCaT cells. Furthermore, shikonin decreased extracellular signal‑regulated kinase (Erk) and Akt phosphorylation. These results indicated that shikonin may exert its anti‑proliferative effects by inducing apoptosis via activation of the mitochondrial signaling pathway and inactivation of the Akt and Erk pathways in HaCaT cells. Therefore, the present study suggested that shikonin may have potential as a component of therapeutic strategies for the treatment of skin diseases.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Extracellular Signal-Regulated MAP Kinases; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Shikonin, which is derived from Lithospermum erythrorhizon, a herb used in traditional medicine, has long been considered to be a useful treatment for various diseases in traditional oriental medicine. Shikonin has recently been reported to have several pharmacological properties, e.g., it has anti-microbial, anti-tumor, and anti-inflammatory effects. The aim of this study was to examine whether shikonin is able to influence the production of interleukin (IL)-6, IL-8, and/or chemokine C-C motif ligand (CCL)20, which contribute to the pathogenesis of periodontal disease, in human periodontal ligament cells (HPDLC). The production levels of IL-6, IL-8, and CCL20 in HPDLC were determined using an ELISA. Western blot analysis was used to detect nuclear factor kappa B (NF-κB) pathway activation in HPDLC. Shikonin prevented IL-1β- or tumor necrosis factor (TNF)-α-mediated IL-6, IL-8, and CCL20 production in HPDLC. Moreover, we found that shikonin suppressed the phosphorylation and degradation of inhibitor of kappa B-alpha (IκB-α) in IL-1β- or TNF-α-stimulated HPDLC. These findings suggest that shikonin could have direct beneficial effects against periodontal disease by reducing IL-6, IL-8, and CCL20 production in periodontal lesions.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Chemokine CCL20; Cytokines; Humans; Inflammation; Interleukin-6; Interleukin-8; Naphthoquinones; Periodontal Diseases; Periodontal Ligament

Shikonin possess a diverse spectrum of pharmacological properties in multiple therapeutic areas. However, the nociceptive effect of shikonin is not largely known. To investigate the antinociceptive potential of shikonin, panel of GPCRs, ion channels, and enzymes involved in pain pathogenesis were studied. To evaluate the translation of shikonin efficacy in vivo, it was tested in 3 established rat pain models. Our study reveals that shikonin has significant inhibitory effect on pan sodium channel/N1E115 and NaV1.7 channel with half maximal inhibitory concentration (IC50) value of 7.6 μmol/L and 6.4 μmol/L, respectively, in a cell-based assay. Shikonin exerted significant dose dependent antinociceptive activity at doses of 0.08%, 0.05%, and 0.02% w/v in pinch pain model. In mechanical hyperalgesia model, dose of 10 and 3 mg/kg (intraperitoneal) produced dose-dependent analgesia and showed 67% and 35% reversal of hyperalgesia respectively at 0.5 h. Following oral administration, it showed 39% reversal at 30 mg/kg dose. When tested in first phase of formalin induced pain, shikonin at 10 mg/kg dose inhibited paw flinching by ∼71%. In all studied preclinical models, analgesic effect was similar or better than standard analgesic drugs. The present study unveils the mechanistic role of shikonin on pain modulation, predominantly via sodium channel modulation, suggesting that shikonin could be developed as a potential pain blocker.

Topics: Analgesics; Animals; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Male; Naphthoquinones; Pain Measurement; Rats; Rats, Sprague-Dawley

The phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation.. The mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production.. The results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.

Topics: Gene Expression Regulation, Plant; Lithospermum; Naphthoquinones; Plant Proteins; Plant Roots; Plants, Genetically Modified; RNA Interference; Transcription Factors

Immunogenic cell death (ICD) of tumor cells occurs via various pathways that activate immune cell systems against cancer. Previous studies have demonstrated that shikonin (SK), a plant secondary metabolite, can confer strong pharmacological activities that activate ICD and strong immunogenicity of tumor cells. However, the exact hierarchical regulatory mechanisms including the molecular targets of SK-activated immunogenicity are still unknown. Here, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was revealed to serve as a specific protein target for SK. This binding plays a key role in SK-stimulated ICD activity and the suppression of post-transcriptional mRNA processing, including nuclear export activity of newly synthesized mRNAs in mammary carcinoma cells in vitro. Moreover, it also mechanistically mediates the anti-metastatic effect of a tumor cell lysate (TCL) vaccine, which can be readily generated from SK-treated 4T1 tumor cells (SK-TCL), and the derived tumor-immunogenicity of SK-TCL-treated dendritic cells in vivo. Together, the identification of hnRNPA1 as the intracellular molecular target provides compelling pharmacology-based knowledge for the potential clinical use of SK-induced immunogenicity. In addition, SK may also serve as a potent suppressor that interferes with specific post-transcriptional activities, a mechanism which may be useful for exploitation in cancer therapeutics.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cancer Vaccines; Cell Line, Tumor; Dendritic Cells; Female; Heterogeneous Nuclear Ribonucleoprotein A1; Humans; Mice; Mice, Inbred BALB C; Molecular Docking Simulation; Naphthoquinones; RNA Processing, Post-Transcriptional; RNA, Messenger; Tandem Mass Spectrometry; Xenograft Model Antitumor Assays

Macrophages play pivotal roles in inflammatory responses. Previous studies showed that various natural products exert antiinflammatory effects by regulating macrophage activation. Recent studies have shown that shikonin (SHK) and its derivatives (β-hydroxyisovalerylshikonin, acetylshikonin, and isobutylshikonin), which are 1,4-naphthoquinone pigments extracted from the roots of Lithospermum erythrorhizon, have various pharmacological, including antiinflammatory and antitumor, effects. Even though there have been many studies on the antiinflammatory activities of SHK derivatives, only a few have described their direct effects on macrophages. We investigated the effects of SHK derivatives on lipopolysaccharide (LPS)-treated macrophages. Low doses of SHK derivatives induced significant macrophage cytotoxicity (mouse macrophage-like J774.1/JA-4 cells and mouse peritoneal macrophages) in the presence of LPS. SHK activated caspases-3 and -7, which led to DNA fragmentation, but this cytotoxicity was prevented through a pan-caspase inhibitor in LPS-treated JA-4 cells. Maximal cytotoxic effects were achieved when SHK was added immediately before LPS addition. These results indicate that SHK derivatives induce caspase-dependent apoptotic cell death of LPS-treated macrophages and suggest that SHK acts during an early stage of LPS signaling.

Topics: Animals; Apoptosis; Caspase 3; Caspase 7; Cell Line; Cells, Cultured; DNA Fragmentation; Female; Lipopolysaccharides; Macrophages; Mice, Inbred BALB C; Naphthoquinones

Naphthoquinones are important class of molecules found as a natural red color pigments in roots of Arnebia benthamii (Wall. ex G. Don) L M. Johnston. The aim of present investigation is to develop and validates a simple, cost-effective and reliable method for quantification of these compounds. Therefore, a normal phase-high performance thin-layer chromatography (NP-HPTLC) method for concurrent determination of shikonin and β,β-dimethylacryl shikonin in A. benthamii was established. Method development of naphthoquinones in the methanol extract was done using hexane-ethyl acetate-methanol (40:7.5:2.5, v/v/v) solvent system at 520 nm. The developed method showed good band separation for shikonin (Rf, 0.37) and β,β-dimethylacryl shikonin (Rf, 0.58). The linearity ranged between 100 and 8,000 ng spot(-1) with an average recovery of >97% in both cases. The results showed reproducible intraday and interday precision (<2.0% RSD) in quantification of naphthoquinones. The limits of detection are 12.96 and 14.65 ng spot(-1) while the limits of quantification are 39.27 and 44.39 ng spot(-1) for shikonin and β,β-dimethylacryl shikonin, respectively. The developed method is reliable, fast, easy to follow and economic in concurrent assessment of shikonin and β,β-dimethylacryl shikonin in A. benthamii root samples. In addition, it seems to be first report for identification and quantification of β,β-dimethylacryl shikonin from the A. benthamii.

Topics: Boraginaceae; Chemistry Techniques, Analytical; Chromatography, Thin Layer; Limit of Detection; Naphthoquinones; Reproducibility of Results; Solvents

Shikonin, the predominant naphthoquinone pigment isolated from the Chinese plant Lithospermum erythrorhizon, is anti‑inflammatory, antiviral and exerts anticancer effects, amongst other biological activities. However, it is unknown whether shikonin affects bone formation. In the present study, the role of shikonin on cell proliferation was assessed via MTT assay, and shikonin was identified to markedly promote cell growth in a time‑ and dose‑dependent manner in the MC3T3‑E1 cell line. In addition, flow cytometric analysis was performed to evaluate the effect of shikonin on the cell cycle, and it was observed that shikonin markedly increased the percentage of S‑phase MC3T3‑E1 cells to accelerate the G1/S transition. To investigate the potential molecular mechanism by which shikonin enhances bone formation, the changes in bone morphogenic protein‑2 (BMP‑2), SMAD family member 5 (Smad5), runt related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC) expression levels induced by shikonin were investigated using western blot analysis and quantitative polymerase chain reaction. The results indicated that shikonin increased the BMP‑2 and Smad5 mRNA levels, and upregulated Smad5 and Runx2 protein expression levels to promote osteoblast differentiation. Furthermore, ALP staining was performed, and revealed that shikonin enhanced ALP activity. These results indicate that shikonin promotes cell proliferation and differentiation of MC3T3-E1 cells via the BMP-2/Smad5 signaling pathway.

Topics: Alkaline Phosphatase; Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomarkers; Bone Morphogenetic Protein 2; Cell Cycle; Cell Differentiation; Cell Line; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Dose-Response Relationship, Drug; Gene Expression; Mice; Naphthoquinones; Signal Transduction; Smad5 Protein

Shikonin possesses anti-inflammatory effects. However, its function in concanavalin A-induced acute liver injury remains uncertain. The aim of the present study was to investigate the functions of shikonin and its mechanism of protection on ConA-induced acute liver injury.. Balb/C mice were exposed to ConA (20 mg/kg) via tail vein injection to establish acute liver injury; shikonin (7.5 mg/kg and 12.5 mg/kg) was intraperitoneally administered 2 h before the ConA injection. The serum liver enzyme levels and the inflammatory cytokine levels were determined at 3, 6, and 24 h after ConA injection.. After the injection of ConA, inflammatory cytokines IL-1β, TNF-α, and IFN-γ were significantly increased. Shikonin significantly ameliorated liver injury and histopathological changes and suppressed the release of inflammatory cytokines. The expressions of Bcl-2 and Bax were markedly affected by shikonin pretreatment. LC3, Beclin-1, and p-JNK expression levels were decreased in the shikonin-pretreated groups compared with the ConA-treated groups. Shikonin attenuated ConA-induced liver injury by reducing apoptosis and autophagy through the inhibition of the JNK pathway.. Our results indicated that shikonin pretreatment attenuates ConA-induced acute liver injury by inhibiting apoptosis and autophagy through the suppression of the JNK pathway.

Topics: Animals; Blotting, Western; Chemical and Drug Induced Liver Injury; Concanavalin A; Immunohistochemistry; Interferon-gamma; Interleukin-1beta; Liver; Male; MAP Kinase Signaling System; Mice, Inbred BALB C; Naphthoquinones; Random Allocation; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Shikonin is a naphthoquinone isolated from the traditional Chinese medicine Lithospermum. It has been used in the treatment of various tumors. However, the effects of shikonin on such diseases have not been fully elucidated. In the present study, we detected the exosome release of a breast cancer cell line (MCF-7) with shikonin treatment and found a positive relationship between the level of secreted exosomes and cell proliferation. We next analyzed miRNA profiles in MCF-7 cells and exosomes and found that some miRNAs are specifically sorted and abundant in exosomes. Knockdown of the most abundant miRNAs in exosomes and the MCF-7 proliferation assay showed that miR-128 in exosomes negatively regulates the level of Bax in MCF-7 recipient cells and inhibits cell proliferation. These results show that shikonin inhibits the proliferation of MCF-7 cells through reducing tumor-derived exosomal miR-128. The current study suggests that shikonin suppresses MCF-7 growth by the inhibition of exosome release.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; Exosomes; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs; Naphthoquinones

ER-Stress and activation of unfolded protein response belong to the major factors involved in chemoresistance in cancer cells. In this study we investigated the effect of shikonin on the survival of acute myeloid leukemia cells and the role of ER-stress protein ERP57, a protein disulfide isomerase, in improvement of chemotherapy.. Using MTT assay we studied cytotoxic effects of shikonin on HL-60 cells. The flow cytometry was adopted to examine the shikonin induced mode of cell death in HL-60 cells. The overall protein expression alteration resulting from shikonin treatment was investigated using proteomics methods. Western blotting was performed to quantify the alteration in protein expression in HL-60 after shikonin treatment. Silencing and overexpression studies were carried out to highlight the therapeutic role of ERP57 in shikonin effect on AML cells.. Shikonin induces apoptosis in HL-60 cells without significant effect on Primary cells from healthy volunteers. The apoptotic effect was dose and time dependent and was accompanied by strong alteration in cell proteome. Among the proteins targeted by shikonin, ERP57 was significantly downregulated in HL-60 after treatment. Compared to healthy control ERP57 was found to be highly expressed in AML cell line HL60 and was downregulated after shikonin treatment. Overexpression of ERP57 protected HL-60 from shikonin induced apoptosis, whereas knockdown of ERP57 expression resulted in increase in shikonin induced apoptosis.. Our results demonstrate that ERP57 plays a crucial role in resistance towards shikonin induced apoptosis in AML cells. Targeting of ERP57 might offer a new therapeutic option for the treatment of acute myeloid leukemia.

Topics: Acute Disease; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Survival; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Electrophoresis, Gel, Two-Dimensional; Endoplasmic Reticulum Stress; Flow Cytometry; HL-60 Cells; Humans; Leukemia, Myeloid; Molecular Structure; Naphthoquinones; Protein Disulfide-Isomerases; Proteome; Proteomics; RNA Interference; Time Factors; Tunicamycin

Topics: A549 Cells; Acetylation; Animals; Antineoplastic Agents; Apoptosis; Bcl-2-Like Protein 11; Carcinoma, Non-Small-Cell Lung; Dose-Response Relationship, Drug; E1A-Associated p300 Protein; Early Growth Response Protein 1; Female; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Sirtuin 1; Time Factors; Transfection; Xenograft Model Antitumor Assays

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.

Topics: Animals; Caspase 1; Caspase Inhibitors; Cell Line, Transformed; DNA-Binding Proteins; Inflammasomes; Interleukin-1beta; L-Lactate Dehydrogenase; Mice; Mice, Inbred C57BL; Naphthoquinones; Nigericin; NLR Family, Pyrin Domain-Containing 3 Protein

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. Elevated levels of PAI-1 are associated with thrombosis and cardiovascular and metabolic diseases. Inhibition of PAI-1 activity represents a new strategy for antithrombotic and antifibrinolysis therapies. In this study, we systematically investigated the inhibitory effect of shikonin on PAI-1 activity. In the chromogenic substrate-based urokinase (uPA)/PAI-1 assay, we found that shikonin inhibited human PAI-1 activity with IC50 values of 30.68±2.32μM. This result was further confirmed by urokinase-type plasminogen activator (uPA)-mediated clot lysis assay. Mechanistic studies indicated that shikonin directly could bind to PAI-1 and prevent the binding of PAI-1 to uPA in a dose-dependent manner. Shikonin also blocked the formation of PAI-1/uPA complex, as shown by SDS/PAGE analysis. In the mouse arterial thrombosis model, intraperitoneal injection of shikonin at 1mgkg(-1) dose significantly prolonged tail bleeding time from 12.956±4.457min to 26.576±2.443min. It also reduced arterial thrombus weight from 0.01±0.001g to 0.006±0.001g (p<0.05). In a liver fibrosis treatment model, when shikonin was continuously injected intraperitoneally at a dose of 1mgkg(-1) over a two-week period, the hydroxyproline content in the mice plasma was significantly reduced and the degree of liver fibrosis was decreased significantly. Thus, shikonin may represent a novel small molecule inhibitor of PAI-1 that could have become a lead drug the treatment of thrombus and fibrosis.

Topics: Animals; Disease Models, Animal; Humans; Liver Cirrhosis; Mice; Naphthoquinones; Plasminogen Activator Inhibitor 1; Thrombosis; Urokinase-Type Plasminogen Activator

The aim of the present study was to investigate the role of nitric oxide (NO) in the antifungal activity of Shikonin (SK) against Candida albicans (C. albicans) and to clarify the underlying mechanism. The results showed that the NO donors S-nitrosoglutathione (GSNO) and L-arginine could enhance the antifungal activity of SK, whereas the NO production inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) attenuated antifungal action. Using the fluorescent dye 3-amino,4-aminomethyl-2', 7-difluorescein, diacetate (DAF-FM DA), we found that the accumulation of NO in C. albicans was increased markedly by SK in a time- and dose-dependent manner. In addition, the results of real-time reverse transcription-PCR (RT-PCR) demonstrated that the transcription level of YHB1 in C. albicans was greatly increased upon incubation of SK. Consistently, the YHB1-null mutant (yhb1Δ/Δ) exhibited a higher susceptibility to SK than wild-type cells. In addition, although the transcription level of CTA4 in C. albicans was not significantly changed when exposed to SK, the CTA4-null mutant (cta4Δ/Δ) was more susceptible to SK. Collectively, SK is the agent found to execute its antifungal activity directly via endogenous NO accumulation, and NO-mediated damage is related to the suppression of YHB1 and the function of CTA4.

Topics: Antifungal Agents; Arginine; Candida albicans; Enzyme Inhibitors; Fungal Proteins; Gene Expression Regulation, Fungal; Microbial Sensitivity Tests; Naphthoquinones; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Real-Time Polymerase Chain Reaction; S-Nitrosoglutathione

Hepatocellular carcinoma (HCC) is a highly lethal malignancy mostly because of metastasis, recurrence and acquired resistance to conventional chemotherapy. Arsenic trioxide (ATO) is successfully used to treat hematological malignancies, and has been proven to trigger apoptosis in HCC cells. However, the phase II trial evaluating the efficacy and toxicity of ATO in patients with HCC showed that single-agent ATO is poorly active against HCC. Therefore, it is of great importance to develop effective chemosensitization agents to ATO. The aim of the present study was to determine whether shikonin (SHI), a natural product from the root of lithospermum erythrorhizon, could synergistically enhance the anti-HCC efficacy of ATO both in vitro and in vivo. We found that the combination of SHI and ATO exhibited synergistic anticancer efficacy and achieved greater selectivity between cancer cells and normal cells. By inducing intracellular oxidative stress, SHI potentiated ATO-induced DNA damage, followed by increased activation of endoplasmic reticulum stress. In addition, inhibition of ROS reversed the apoptosis induced by SHI and ATO, and recovered the activation of endoplasmic reticulum stress, which revealed the vital role of ROS in the synergism. Moreover, HepG2 xenograft tumor growth in nude mice was more effectively inhibited by combined treatment with SHI and ATO. These data suggest that the combination of SHI with ATO presents a promising therapeutic approach for the treatment of HCC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arsenic Trioxide; Arsenicals; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; DNA Damage; Drug Synergism; Endoplasmic Reticulum Stress; Hep G2 Cells; Humans; Liver Neoplasms; Mice, Nude; Naphthoquinones; Oxides; Xenograft Model Antitumor Assays

Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2-EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis.

Topics: Animals; Carrier Proteins; Coinfection; Disease Models, Animal; DNA-Binding Proteins; eIF-2 Kinase; Female; Glycolysis; HMGB1 Protein; Humans; Inflammasomes; Interleukin-18; Interleukin-1beta; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Myeloid Cells; Naphthoquinones; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphorylation; Pyruvate Kinase; Sepsis; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones

The general mechanism underlying the tumor suppressor activity of the Hippo signaling pathway remains unclear. In this study, we explore the molecular mechanisms connecting the Hippo signaling pathway with glucose metabolism. We have found that two key regulators of glycolysis, C-MYC and GLUT1, are targets of the Hippo signaling pathway in human leukemia cells. Our results revealed that activation of MST1 by the natural compound shikonin inhibited the expression of GLUT1 and C-MYC. Furthermore, RNAi experiments confirmed the regulation of GLUT1 and C-MYC expression via the MST1-YAP1-TEAD1 axis. Surprisingly, YAP1 was found to positively regulate C-MYC mRNA levels in complex with TEAD1, while it negatively regulates C-MYC levels in cooperation with MST1. Hence, YAP1 serves as a rheostat for C-MYC, which is regulated by MST1. In addition, depletion of MST1 stimulates lactate production, whereas the specific depletion of TEAD1 has an opposite effect. The inhibition of lactate production and cellular proliferation induced by shikonin also depends on the Hippo pathway activity. Finally, a bioinformatic analysis revealed conserved TEAD-binding motifs in the C-MYC and GLUT1 promoters providing another molecular data supporting our observations. In summary, regulation of glucose metabolism could serve as a new tumor suppressor mechanism orchestrated by the Hippo signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Cell Proliferation; DNA-Binding Proteins; Genes, myc; Glucose Transporter Type 1; Hepatocyte Growth Factor; Humans; Naphthoquinones; Nuclear Proteins; Phosphoproteins; Proto-Oncogene Proteins; Signal Transduction; TEA Domain Transcription Factors; Transcription Factors; YAP-Signaling Proteins

The prognosis of gastric cancer remains poor due to clinical drug resistance. Novel drugs are urgently needed. Shikonin (SHK), a natural naphthoquinone, has been reported to trigger cell death and overcome drug resistance in anti-tumour therapy. In this study, we investigated the effectiveness and molecular mechanisms of SHK in treatment with gastric cancer. In vitro, SHK suppresses proliferation and triggers cell death of gastric cancer cells but leads minor damage to gastric epithelial cells. SHK induces the generation of intracellular reactive oxygen species (ROS), depolarizes the mitochondrial membrane potential (MMP) and ultimately triggers mitochondria-mediated apoptosis. We confirmed that SHK induces apoptosis of gastric cancer cells not only in a caspase-dependent manner which releases Cytochrome C and triggers the caspase cascade, but also in a caspase-independent manner which mediates the nuclear translocation of apoptosis-inducing factor and Endonuclease G. Furthermore, we demonstrated that SHK enhanced the chemotherapeutic sensitivity of 5-fluorouracil and oxaliplatin in vitro and in vivo. Taken together, our data show that SHK may be a novel therapeutic agent in the clinical treatment of gastric cancer.

Topics: Apoptosis; Cell Line, Tumor; Fluorouracil; Humans; Mitochondria; Naphthoquinones; Organoplatinum Compounds; Pyridines; Reactive Oxygen Species; Stomach Neoplasms

The antitumor activity of shikonin derivatives is largely dependent on the generation of superoxide radicals and the alkylation activity of their naphthoquinone moiety. However, our recent study showed that 1,4-dioxime-5,8-dimethoxynaphthalene (DMAKO-05), a novel shikonin derivative, displayed more potential antitumor activity and less toxicity compared to fluorouracil (5-FU) both in vitro and in vivo, even though the hydroxyl and carbonyl groups of its naphthoquinone structure were shielded. To understand the underlying mechanisms, we investigated the metabolism of DMAKO-05 in rat liver microsomes. The kinetic analysis indicated that DMAKO-05 underwent a biphasic metabolism in rat liver microsomes. The inhibition experiments showed that CYP1A and CYP3A were the major enzymes in the metabolism of DMAKO-05, along with partial contribution from CYP2A. In addition, the structures of eight DMAKO-05 metabolites, which were characterized by accurate mass and MS/MS fragmentograms, implied that DMAKO-05 was mainly metabolized through the oxygenation of its naphthoquinone nucleus and the hydrolysis of its side chain, instead of the oxidation of hydroxyimine to ketone. Therefore, DMAKO-05 should not be considered as a traditional naphthoquinone prodrug.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Fluorouracil; HCT116 Cells; Humans; Kinetics; Male; Microsomes, Liver; Naphthoquinones; Prodrugs; Rats; Rats, Sprague-Dawley

Overexpression of SIRT1 is considered to enhance the resistance of HepG2 cells to irradiation. Shikonin, a naturally occurring naphthoquinone compound, displays anticancer effects and circumvents cancer drug resistance.. This study investigated the MDR reversal effect of shikonin induced by the overexpression of SIRT1.. The overexpression of SIRT1 in HepG2 cells was established by lentivirus infection. Five days after transduction, real-time quantitative polymerase chain reaction and western blotting were used to detect the expression of SIRT1 and MDR1/P-gp. Drug resistance was also evaluated by flow cytometry after rhodamine-123 staining. On day 5, the multidrug resistance cells were treated by shikonin (10(-7), 10(-6), and 10(-5) µmol/L) one time. The cell viability was detected by the MTT assay, and apoptosis was evaluated by Hoechst 33342 staining and caspase-3 activity 24 h after shikonin treatment.. Overexpression of SIRT1 decreased rhodamine-123 staining and successfully produced the R-HepG2 cell line. Compared with HepG2, the expression of MDR1/P-gp mRNA (3.45 ± 0.35) and protein (1.40 ± 0.05) were both upregulated in R-HepG2. Shikonin inhibited cell viability (from 93.9 ± 2.1 to 66.7 ± 1.5%), induced apoptosis of R-HepG2 (apoptotic ratio from 3.5 ± 0.8 to 47.5 ± 2.7%, caspase-3 activity from 103.5 ± 1.9 to 329.2 ± 14.9%, respectively), downregulated the mRNA and protein expression of SIRT1 and MDR1/P-gp, and decreased rhodamin 123 efflux.. In the present study, we demonstrated that shikonin is able to overcome drug resistance in hepatocellular carcinoma cells, and the mechanism is related to the SIRT1-MDR1/P-gp signaling pathway.

Topics: Cell Survival; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Hep G2 Cells; Humans; Naphthoquinones; Sirtuin 1

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5-5 µM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 µM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10-20 µM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Death; Cell Line, Tumor; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Humans; Imidazoles; Indoles; Multiple Myeloma; Naphthoquinones; Necrosis; Proteasome Inhibitors; Purine Nucleosides; Pyrazines; Regulatory Factor X Transcription Factors; Transcription Factors; X-Box Binding Protein 1

Oxidized low-density lipoprotein (oxLDL) is a key contributor to atherogenesis through multiple mechanisms, including the reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NFκB) signaling pathway. Although shikonin, one of the main active components isolated from the Chinese herb Lithospermum erythrorhizon, has been shown to possess cardioprotective, antioxidative, and anti-inflammatory effects, the mechanisms underlying these actions are not well understood. In this study, we used EA.hy926 endothelial-like cells to examine the anti-atherogenic activity of shikonin. Shikonin (0-1 μM) concentration-dependently induced heme oxygenase-1, glutamate cysteine ligase modifier subunit, catalase, superoxide dismutase 1, glutathione peroxidase 1, and glutathione reductase protein and mRNA expression and glutathione content via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/Nrf2 signaling pathway. In the presence of oxLDL (40 μg/ml), shikonin pretreatment reversed oxLDL-induced ROS production, antioxidant response element reporter activity, NFκB nuclear translocation, and intercellular adhesion molecule (ICAM)-1 and E-selectin expression and suppressed the increase of monocyte adhesion to endothelial cells. Nrf2 knockdown by using RNA interference attenuated the ability of shikonin to inhibit oxLDL-induced NFκB DNA binding activity, adhesion molecule expression, and monocyte adhesion. Taken together, these results suggest that shikonin protects against oxLDL-induced endothelial damage by suppressing ROS/NFκB-mediated ICAM-1 and E-selectin expression via up-regulation of PI3K/Akt/Nrf2-dependent antioxidant enzyme expression.

Topics: Antioxidants; Cell Adhesion; Cell Line, Tumor; Dose-Response Relationship, Drug; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins, LDL; Monocytes; Naphthoquinones; NF-E2-Related Factor 2; NF-kappa B; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Up-Regulation

Root bark of Onosma echioides belonging to the family Boraginaceae is reported to be rich in naphthaquinones such as alkannins and shikonins. In this study, a dimer of alkannin/shikonin was isolated from the petroleum ether (60-80 C) extract of the bark, and the structure of the same was elucidated through spectral studies (UV, IR, NMR, MS and DEPT). The petroleum ether extract was found to contain 62.4% (w/w) of the dimer of alkannin/shikonin, and the compound is found to promote wound-healing process, when studied in the excision and incision wound models in albino rats.

Topics: Animals; Boraginaceae; Molecular Structure; Naphthoquinones; Plant Extracts; Plant Roots; Rats; Wound Healing

Lithospermum erythrorhizon has been proved to be anti-inflammatory, by recent studies. This study extracts L. erythrorhizon with ethanol at various solid-liquid ratios (1:4, 1:6, 1:8, and 1:12), extraction temperatures (40°C, 50°C, and 60°C), and extraction times (4, 24 and 36h) in order to determine the optimal parameters. The optimal parameters are extracted and condensed into L. erythrorhizon extract; then the antibacterial property and cell compatibility of L. erythrorhizon extract are evaluated with various concentrations of L. erythrorhizon extract solution and different weights of L. erythrorhizon extract powder, respectively. The concentrations of solution are 0.1mg/ml, 0.5mg/ml, 1.0mg/ml, and 2.0mg/ml and ethanol is chosen as the solvent, and different weights of powder are varied as 0.1mg, 1.0mg, 2.0mg, and 10mg. The cell viability test and animal study are performed on L. erythrorhizon microcapsules. The experiment results show that sodium alginate/pectin L. erythrorhizon (SPL) microcapsules possess a 120-hour drug release. The results of cell viability and animal study show that the L. erythrorhizon microcapsules (SPL) have good cell viability (99%) and can help in the wound healing process (the wound size reduction reaches 91.3% on Day 11).

Topics: Alginates; Animals; Anti-Bacterial Agents; Capsules; Cell Survival; Drug Delivery Systems; Glucuronic Acid; Hexuronic Acids; Lithospermum; Male; Naphthoquinones; Pectins; Plant Extracts; Powders; Rats, Sprague-Dawley; Staphylococcus aureus; Wound Healing

Scarring is a significant medical burden; financially to the health care system and physically and psychologically for patients. Importantly, there have been numerous case reports describing the occurrence of cancer in burn scars. Currently available therapies are not satisfactory due to their undesirable side-effects, complex delivery routes, requirements for long-term use and/or expense. Radix Arnebiae (Zi Cao), a perennial herb, has been clinically applied to treat burns and manage scars for thousands of years in Asia. Shikonin, an active component extracted from Radix Arnebiae, has been demonstrated to induce apoptosis in cancer cells. Apoptosis is an essential process during scar tissue remodelling. It was therefore hypothesized that Shikonin may induce apoptosis in scar-associated cells. This investigation presents the first detailed in vitro study examining the functional responses of scar-associated cells to Shikonin, and investigates the mechanisms underlying these responses. The data obtained suggests that Shikonin inhibits cell viability and proliferation and reduces detectable collagen in scar-derived fibroblasts. Further investigation revealed that Shikonin induces apoptosis in scar fibroblasts by differentially regulating the expression of caspase 3, Bcl-2, phospho-Erk1/2 and phospho-p38. In addition, Shikonin down-regulates the expression of collagen I, collagen III and alpha-smooth muscle actin genes hence attenuating collagen synthesis in scar-derived fibroblasts. In summary, it is demonstrated that Shikonin induces apoptosis and decreases collagen production in scar-associated fibroblasts and may therefore hold potential as a novel scar remediation therapy.

Topics: Apoptosis; Cell Proliferation; Cell Survival; Cells, Cultured; Cicatrix; Collagen; Dose-Response Relationship, Drug; Humans; Keratinocytes; Naphthoquinones; Structure-Activity Relationship

In this study, a shikonin ester derivative, compound , was selected to evaluate its anticancer activities and we found that compound exhibited better antitubulin activities against the human HepG2 cell line with an IC50 value of 1.097 μM. Furthermore, the inhibition of tubulin polymerization results indicated that compound demonstrated the most potent antitubulin activity (IC50  = 13.88), which was compared with shikonin and colchicine as positive controls (IC50  = 25.28 μM and 22.56 μM), respectively. Compound was simulated to have good binding site with tubulin and arrested the cell cycle at G2/M phase, which also induces apoptosis in HepG2 cells, in which P53 and members of Bcl-2 protein family were both involved in the progress of apoptosis revealed by western blot. Confocal microscopy observations revealed compound targeted tubulin and altered its polymerization by interfering with microtubule organization. Based on these results, compound functions as a potent anticancer agent targeting tubulin.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Humans; Inhibitory Concentration 50; Microtubules; Molecular Docking Simulation; Naphthoquinones; Tubulin Modulators

Glucose-regulated protein 78 (GRP78) is a key modulator of prostate cancer progression and therapeutic resistance. Prostate cancer is a worldwide health problem, and therapeutic resistance is a critical obstacle for the treatment of hormone-refractory prostate cancer patients. Shikonin inhibits prostate cancer proliferation and metastasis. However, the role of GRP78 in the cytotoxic effect of shikonin in prostate cancer cells remains unclear. GRP78 expression was abolished using small interfering RNA (siRNA), and the anticancer effects of shikonin were assessed using MTT assays, the XCELLigence biosensor, flow cytometric cell cycle analysis, and Annexin V-PI apoptotic assays. PC-3 cells expressed more GRP78 than DU-145 cells, and the MTT assays revealed that DU-145 cells were more sensitive to shikonin than PC-3 cells. GRP78 knockdown (GRP78KD) PC-3 cells were more sensitive to shikonin treatment than scrambled siRNA control cells. Based on cell cycle analysis and AnnexinV-PI apoptotic assays, apoptosis dramatically increased in GRP78KD cells compared with the control PC-3 in response to shikonin. Finally, in response to shikonin treatment, Mcl-1 and Bcl-2 levels increased in the scrambled control cells treated with shikonin, whereas Bcl-2 decreased and Mcl-1 slightly increased in the GRP78KD PC-3 cells. The levels of Bax and Bad did not change in the scrambled control or GRP78KD cells after shikonin treatment. These results are consistent with the increased sensitivity to shikonin after knockdown of GRP78. GRP78 expression may determine the therapeutic efficacy of shikonin against prostate cancer cells.

Topics: Apoptosis; Biosensing Techniques; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum Chaperone BiP; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Male; Naphthoquinones; Prostatic Neoplasms

Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in tumorigenesis and resistance to treatment of glioblastoma (GBM). EGFR inhibitors such as erlotinib are currently used for the treatment of GBM; however, their efficacy has been limited due to drug resistance. New treatment strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and necroptosis in human glioma cells, but the effectiveness of erlotinib-shikonin combination treatment as well as the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.ΔEGFR GBM cells. Most of the shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely deoxyshikonin, isobutyrylshikonin, acetylshikonin, β,β-dimethylacrylshikonin and acetylalkannin showed synergistic cytotoxicity toward U87MG.ΔEGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of shikonin and erlotinib was further confirmed with another three EGFR-expressing cell lines, BS153, A431 and DK-MG. Shikonin not only dose-dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42MAPK and PLCγ1, but also together with erlotinib synergistically inhibited ΔEGFR phosphorylation in U87MG.ΔEGFR cells as determined by Loewe additivity and Bliss independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its derivatives might be a potential strategy to overcome drug resistance to erlotinib.

Topics: Anthraquinones; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; Glioblastoma; Humans; Mitogen-Activated Protein Kinases; Naphthoquinones; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction

Radiation encephalopathy is the main complication of cranial radiotherapy. It can cause necrosis of brain tissue and cognitive dysfunction. Our previous work had proved that a natural antioxidant shikonin possessed protective effect on cerebral ischemic injury. Here we investigated the effects of shikonin on carbon ion beam induced radiation brain injury in mice. Pretreatment with shikonin significantly increased the SOD and CAT activities and the ratio of GSH/GSSG in mouse brain tissues compared with irradiated group (P<0.01), while obviously reduced the MDA and PCO contents and the ROS levels derived from of the brain mitochondria. The shikonin also noticeably improved the spatial memory deficits caused by carbon ion beam irradiation. All results demonstrated that shikonin could improve the irradiated brain injury which might resulted from its modulation effects on the oxidative stress induced by the 12C6+ ion beam.

Topics: Animals; Antioxidants; Brain Injuries; Catalase; Heavy Ion Radiotherapy; Male; Malondialdehyde; Mice; Naphthoquinones; Protein Carbonylation; Radiation Injuries, Experimental; Radiation-Protective Agents; Random Allocation; Specific Pathogen-Free Organisms; Superoxide Dismutase

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal Zicao, has been shown to exhibit antioxidant and anticancer effects. In the present study, we investigated the antiproliferative and pro-apoptotic effects of shikonin on A431 cells and explored the underlying molecular mechanisms. In the present study, our results showed that shikonin significantly inhibited the growth of A431 cells in a concentration- and time-dependent manner, and caused cell cycle arrest by upregulation of p21 and p27, and downregulation of cyclins and cyclin-dependent kinases. In addition, shikonin evidently induced apoptosis due to decreasing Bcl-2 expression, increasing Bax expression, activating caspase and inactivating NF-κB, while pretreatment with a pan-caspase inhibitor Z-Asp-CH2-DCB abrogated shikonin-induced apoptosis. Moreover, EGF could significantly increase the NF-κB DNA-binding activity and reversed the shikonin-induced inactivation of NF-κB. As anticipated AG1478 (EGFR inhibitor) and Bay11-7082 (NF-κB inhibitor) blocked EGF-reversed the inactivation of NF-κB induced by shikonin. Our data also showed that EGF could evidently reverse the shikonin-induced decreases in cell viability and increases in apoptosis. Then, the NF-κB inhibitors such as Bay11-7082, SN50, Helenalin and the EGFR inhibitor AG1478 and its downstream inhibitor such as PI3K inhibitor LY294002 and STAT3 inhibitor Stattic dramatically blocked EGF-reversed decreases in cell viability and increases in apoptosis induced by shikonin. Collectively, our findings indicated that shikonin inhibited cell growth and caused cell cycle arrest of the A431 cells through the regulation of apoptosis. Moreover, these effects were mediated at least partially by suppressing the activation of the EGFR-NF-κB signaling pathways.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Dose-Response Relationship, Drug; ErbB Receptors; Humans; Naphthoquinones; Neoplasm Proteins; NF-kappa B

Chondrocyte apoptosis is mostly responsible for the development and progression of osteoarthritis. IL-1β is generally served as an agent that induces chondrocyte apoptosis. Shikonin exerts its anti-inflammatory effect on cartilage protection in vivo. We aimed to explore the protective effect of shikonin on interleukin-1beta (IL-1β)-induced chondrocyte apoptosis and the potential molecular mechanisms. Chondrocytes were isolated from the joints of newborn Sprague-Dawley rats. The MTT assay and LDH cell death assay were used to determine the cell viability and chondrocyte apoptosis was detected by Annexin-V/PI staining and nucleosomal degradation. The contents of phosphorylated-PI3K (p-PI3k), phosphorylated-Akt (p-Akt), Bcl-2, Bax, and cytochrome c were detected by Western blotting. A quantitative colorimetric assay was used to detect the caspase-3 activity. Our results showed that pretreatment with shikonin (4 μM) inhibited cytotoxicity and apoptosis induced by IL-1β (10 ng/ml) in chondrocytes. Shikonin pretreatment also decreased the activity of IL-1β that decreased Bcl-2 expression and levels of p-PI3K and p-Akt, and increased Bax expression, cytochrome c release, and caspase-3 activation. It also reversed the activity of IL-1β that promoted the synthesis of matrix metalloproteinase-13 and inhibited the expression of tissue inhibitor of metalloproteinase-1 expression, with the net effect of suppressing extracellular matrix degradation. These data suggested that shikonin may protect chondrocytes from apoptosis induced by IL-1β through the PI3K/Akt signaling pathway, by deactivating caspase-3.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Western; Cell Survival; Cells, Cultured; Chondrocytes; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Male; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

The tumor microenvironment plays a critical role in regulating breast tumor progression. Signaling between preadipocytes and breast cancer cells has been found to promote breast tumor formation and metastasis. Exosomes secreted from preadipocytes are important components of the cancer stem cell niche. Mouse preadipocytes (3T3L1) are treated with the natural antitumor compound shikonin (SK) and exosomes derived from mouse preadipocytes are co-cultured with MCF10DCIS cells. We examine how preadipocyte-derived exosomes can regulate early-stage breast cancer via regulating stem cell renewal, cell migration, and tumor formation. We identify a critical miR-140/SOX2/SOX9 axis that regulates differentiation, stemness, and migration in the tumor microenvironment. Next, we find that the natural antitumor compound SK can inhibit preadipocyte signaling inhibiting nearby ductal carcinoma in situ (DCIS) cells. Through co-culture experiments, we find that SK-treated preadipocytes secrete exosomes with high levels of miR-140, which can impact nearby DCIS cells through targeting SOX9 signaling. Finally, we find that preadipocyte-derived exosomes promote tumorigenesis in vivo, providing strong support for the importance of exosomal signaling in the tumor microenvironment. Our data also show that targeting the tumor microenvironment may assist in blocking tumor progression.

Topics: 3T3 Cells; Adipocytes; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Exosomes; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Naphthoquinones; Neoplasm Transplantation; Neoplastic Stem Cells; Signal Transduction; SOX9 Transcription Factor

Shikonin, a naturally occurring naphthoquinone, exhibits anti-tumorigenic activity. However, its precise mechanisms of action have remained elusive. In the present study, the involvement in the action of shikonin of the ubiquitin ligases Cbl-b and c-Cbl, which are negative regulators of phosphoinositide 3-kinase (PI3K) activation, was investigated. Shikonin was observed to reduce cell viability and induce apoptosis and G2/M phase arrest in lung cancer cells. In addition, shikonin increased the protein levels of B-cell lymphoma 2 (Bcl-2)-associated X and p53 and reduced those of Bcl-2. Additionally, shikonin inhibited PI3k/Akt activity and upregulated Cbl protein expression. In addition, a specific inhibitor of PI3K, LY294002, was observed to have a synergistic effect on the proliferation inhibition and apoptotic induction of A549 cells with shikonin. In conclusion, the results of the present study suggested that Cbl proteins promote shikonin-induced apoptosis by negatively regulating PI3K/Akt signaling in lung cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Chromones; Drug Synergism; Epithelial Cells; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Morpholines; Naphthoquinones; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-cbl; Respiratory Mucosa; Signal Transduction; Tumor Suppressor Protein p53

Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.. The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.. The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum Stress; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Prostatic Neoplasms

Shikonin is an active naphthoquinone pigment isolated from the root of Lithospermum erythrorhizon. This study was designed to explore the inhibition of Shikonin on cell viability, adhesion, migration and invasion ability of gastric cancer (GC) and its possible mechanism.. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed for cell viability and adhesion ability of MGC-803 cells. Cell scratch repair experiments were conducted for the determination of migration ability while transwell assay for cell invasion ability. Western blot analysis and real-time polymerase chain reaction assay were used for the detection of protein and mRNA expressions.. Fifty per cent inhibitory concentration of Shikonin on MGC-803 cells was 1.854 μm. Shikonin (1 μm) inhibited significantly the adhesion, invasion and migratory ability of MGC-803 cells. Interestingly, Shikonin in the presence or absence of anti-Toll-like receptor 2 (TLR2) antibody (2 μg) and nuclear factor-kappa B (NF-κB) inhibitor MG-132 (10 μm) could decrease these ability of MGC-803 cells markedly, as well as the expression levels of matrix metalloproteinases (MMP)-2, MMP-7, TLR2 and p65 NF-κB. In addition, the co-incubation of Shikonin and anti-TLR2/MG-132 has a significant stronger activity than anti-TLR2 or MG-132 alone.. The results indicated that Shikonin could suppress the cell viability, adhesion, invasion and migratory ability of MGC-803 cells through TLR2- or NF-κB-mediated pathway. Our findings provide novel information for the treatment of Shikonin on GC.

Topics: Antineoplastic Agents; Cell Adhesion; Cell Line, Tumor; Cell Survival; Humans; Inhibitory Concentration 50; Lithospermum; Matrix Metalloproteinase 2; Matrix Metalloproteinase 7; Naphthoquinones; NF-kappa B; Real-Time Polymerase Chain Reaction; Stomach Neoplasms; Toll-Like Receptor 2

2-Deoxy-d-glucose (2-DG), a synthetic glycolytic inhibitor, is currently under clinical evaluation as a promising anticancer agent. However, 2-DG treatment in cancer cells activates prosurvival Akt signaling that might limit its clinical efficacy. The NADPH oxidase 4 (Nox-4)/reactive oxygen species/Akt signaling is known to regulate survival, proliferation, infiltration, and invasion in glioblastomas (GBMs). The enhanced motility, invasiveness, and therapy resistance in GBMs are attributed to metabolic adaptation through increased aerobic glycolysis. Therefore, we hypothesized that inhibition of the Nox-4 might enhance 2-DG-induced suppression of glycolysis, migration, and invasion in GBM cells.. We identified the natural naphthoquinone compound shikonin as a potent inhibitor of the Nox-4/Akt signaling pathway. The combined treatment of shikonin+2-DG suppressed the glycolytic phenotype, migration, and invasion through modulation of the Akt/HIF1α/hexokinase-2 signaling axis in GBM cells. The combination also exhibited enhanced antiproliferative and antiangiogenic effects in vivo.. Our data for the first time demonstrate that inhibition of the Nox-4-associated prosurvival signaling pathway by shikonin enhances the antiproliferative and antiangiogenic potential of 2-DG in GBM cells.. In summary, the combined inhibition of Nox-4 and glycolysis may have therapeutic implications for the management of GBMs.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Movement; Deoxyglucose; Enzyme Inhibitors; Glioblastoma; Glycolysis; Humans; NADPH Oxidases; Naphthoquinones; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Signal Transduction

Although cancer stem-like cells (CSCs) are rare, they can enter a non-proliferative or dormant state and resist therapy. Furthermore, quiescent CSCs are responsible for metastases that can appear after curative surgical treatment of a primary tumor. Because of drug resistance of CSCs, the development of novel therapies is urgently required that specifically target CSCs.. The aim of the present study was to investigate the potential of a panel of natural products and derivatives to inhibit CSC-enriched mammospheres of MCF-7 breast cancer cells.. CD44(high)/CD24(low) cells were identified by flow cytometry and maintained as mammospheres. As a control, we used two clinically established anticancer drugs (5-fluorouracil and docetaxel). A panel of natural products, shikonin, two cajanin stilbene acid (CSA) derivatives and artesunate were tested by resazurin assay on CSC-enriched mammospheres and MCF-7 monolayer cells. Besides, cellular shikonin uptake experiments were performed by flow cytometry.. We found two CSA derivatives (Nos. 6 and 19) to be active cancer stem-like MCF-7 mammospheres. Especially, CSA derivative No. 19 clearly showed collateral sensitivity in mammospheres compared to monolayer cells.. Phytochemicals which provoke collateral sensitivity in cancer-stem like cells are worth more detailed investigations in the future, since there is a great potential for improved chemotherapy to eradicate tumors and prolong cancer patients' survival times.

Topics: Antineoplastic Agents; Artemisinins; Artesunate; Humans; MCF-7 Cells; Naphthoquinones; Neoplastic Stem Cells; Phytochemicals; Salicylates; Spheroids, Cellular; Stilbenes

The high incidence of cancer and the side effects of traditional anticancer drugs motivate the search for new and more effective anticancer drugs. In this study, we synthesized 17 kinds of aryl dihydrothiazol acyl shikonin ester derivatives and evaluated their anticancer activity through MTT assay. Among them, C13 showed better antiproliferation activity with IC50=3.14 ± 0.21 μM against HeLa cells than shikonin (IC50=5.75 ± 0.47 μM). We then performed PI staining assay, cell cycle distribution, and cell apoptosis analysis for C13 and found that it can cause cell arrest in G2/M phase, which leads to cell apoptosis. This derivative can also reduce the adhesive ability of HeLa cells. Docking simulation and confocal microscopy assay results further indicated that C13 could bind well to the tubulin at paclitaxel binding site, leading to tubulin polymerization and mitotic disruption.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Cycle Checkpoints; Cell Line; Cell Line, Tumor; Cell Proliferation; Chlorocebus aethiops; Drug Screening Assays, Antitumor; Esters; Humans; Molecular Docking Simulation; Naphthoquinones; Structure-Activity Relationship; Thiazoles; Tubulin; Tubulin Modulators

The M2 isoform of pyruvate kinase M2 (PKM2) has been shown to be up-regulated in human skin cancers. To test whether PKM2 may be a target for chemoprevention, shikonin, a natural product from the root of Lithospermum erythrorhizon and a specific inhibitor of PKM2, was used in a chemically-induced mouse skin carcinogenesis study. The results revealed that shikonin treatment suppressed skin tumor formation. Morphological examinations and immunohistochemical staining of the skin epidermal tissues suggested that shikonin inhibited cell proliferation without inducing apoptosis. Although shikonin alone suppressed PKM2 activity, it did not suppress tumor promoter-induced PKM2 activation in the skin epidermal tissues at the end of the skin carcinogenesis study. To reveal the potential chemopreventive mechanism of shikonin, an antibody microarray analysis was performed, and the results showed that the transcription factor ATF2 and its downstream target Cdk4 were up-regulated by chemical carcinogen treatment; whereas these up-regulations were suppressed by shikonin. In a promotable skin cell model, the nuclear levels of ATF2 were increased during tumor promotion, whereas this increase was inhibited by shikonin. Furthermore, knockdown of ATF2 decreased the expression levels of Cdk4 and Fra-1 (a key subunit of the activator protein 1. In summary, these results suggest that shikonin, rather than inhibiting PKM2 in vivo, suppresses the ATF2 pathway in skin carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Activating Transcription Factor 2; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinogens; Carrier Proteins; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase 4; Epidermis; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; Mice, Inbred DBA; Naphthoquinones; Proto-Oncogene Proteins c-fos; Pyridines; Signal Transduction; Skin Neoplasms; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcriptional Activation

Cancer stem cells (CSCs) are responsible for aggressive tumor growth, metastasis and therapy resistance. In this study, we evaluated the effects of Shikonin (Shk) on breast cancer and found its anti-CSC potential. Shk treatment decreased the expression of various epithelial to mesenchymal transition (EMT) and CSC associated markers. Kinase profiling array and western blot analysis indicated that Shk inhibits STAT3, FAK and Src activation. Inhibition of these signaling proteins using standard inhibitors revealed that STAT3 inhibition affected CSCs properties more significantly than FAK or Src inhibition. We observed a significant decrease in cell migration upon FAK and Src inhibition and decrease in invasion upon inhibition of STAT3, FAK and Src. Combined inhibition of STAT3 with Src or FAK reduced the mammosphere formation, migration and invasion more significantly than the individual inhibitions. These observations indicated that the anti-breast cancer properties of Shk are due to its potential to inhibit multiple signaling proteins. Shk also reduced the activation and expression of STAT3, FAK and Src in vivo and reduced tumorigenicity, growth and metastasis of 4T1 cells. Collectively, this study underscores the translational relevance of using a single inhibitor (Shk) for compromising multiple tumor-associated signaling pathways to check cancer metastasis and stem cell load.

Topics: Adult; Anti-Inflammatory Agents, Non-Steroidal; Breast Neoplasms; Cell Movement; Cell Proliferation; Cell Survival; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Female; Focal Adhesion Kinase 1; Humans; MCF-7 Cells; Middle Aged; Naphthoquinones; Neoplasm Invasiveness; Neoplastic Stem Cells; Proto-Oncogene Proteins pp60(c-src); Signal Transduction; Spheroids, Cellular; STAT3 Transcription Factor; Tumor Burden; Tumor Cells, Cultured

Shikonin is a naphthoquinone compound extracted from the Chinese herb purple gromwell. Shikonin has broad antibacterial, anti-inflammatory, and antitumor activities. The tumor necrosis factor-α (TNF-α)-induced proliferation and invasion of vascular smooth muscle cells (VSMCs) is an important factor that contributes to atherosclerosis. The effects of shikonin on the proliferation and apoptosis of VSMCs have been reported; however, the function of shikonin on TNF-α-mediated growth and invasion of VSMCs during atherosclerosis remains unclear. In this study, we used Western blot, flow cytometry, real-time quantitative PCR, and enzyme-linked immunosorbent assay to investigate the effect of shikonin on the TNF-α-induced growth and invasion of VSMCs and to determine the underlying mechanism. Our results showed that shikonin inhibits the TNF-α-mediated growth and invasion. Further study revealed that shikonin regulates the activation of nuclear factor kappa B and phosphatidyl inositol 3-kinase signaling pathways; modulates the expression of cyclin D1, cyclin E, B-cell lymphoma 2, and Bax; activates caspase-3 and caspase-9; induces cell cycle arrest; and promotes the apoptosis of VSMCs. Together, our results indicate that shikonin may become a promising agent for the treatment of atherosclerosis and they also establish foundation for the development of anti-atherosclerosis drugs.

Topics: Animals; Anti-Inflammatory Agents; Aorta; Apoptosis; Cell Cycle Checkpoints; Cell Movement; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Regulation; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthoquinones; Rats, Sprague-Dawley; Signal Transduction; Tumor Necrosis Factor-alpha

Shikonin, one of the active components isolated from the root of Arnebia euchroma (Royle) Johnst, have anti-tumor, anti-bacterial and anti-inflammatory activities and has been used clinically in phlebitis and vascular purpura. In the present work, the interaction of human immunoglobulin (HIg) with shikonin has been investigated by using scanning electron microscope (SEM), Fourier transform infrared (FT-IR) spectroscopy, fluorescence polarization, synchronous and 3D fluorescence spectroscopy in combination with molecular modeling techniques under physiological conditions with drug concentrations of 3.33-36.67 μM. The results of SEM exhibited visually the special effect on aggregation behavior of the complex formed between HIg and shikonin. The fluorescence polarization values indicated that shikonin molecules were found in a motionally unrestricted environment introduced by HIg. Molecular docking showed the shikonin moiety bound to the hydrophobic cavity of HIg, and there are four hydrogen-bonding interactions between shikonin and the residues of protein. The synchronous and 3D fluorescence spectra confirmed that shikonin could quench the intrinsic fluorescence of HIg and has an effect on the microenvironment around HIg in aqueous solution. The changes in the secondary structure of HIg were estimated by qualitative and quantitative FT-IR spectroscopic analysis. The binding constants and thermodynamic parameters for shikonin-HIg systems were obtained under different temperatures (300 K, 310 K and 320 K). The above results revealed the binding mechanism of shikonin and HIg at the ultrastructure and molecular level.

Topics: Binding Sites; Fluorescence; Humans; Hydrogen Bonding; Immunoglobulins; Microscopy, Electron, Scanning; Models, Molecular; Naphthoquinones; Protein Binding; Protein Structure, Secondary; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Thermodynamics

To observe the effect of shikonin on the proliferation of human keratinocytes induced by IL-17 and secretion of chemokines, in order to discuss the mechanism of Shikonin in the treatment of psoriasis.. In vitro cultured HaCaT cells were stimulated by IL-17A (200 μg x L(-1)) and mixed with different concentrations (2, 1 mg x L(-1)) of shikonin for 24 hours. The cell proliferation was detected by CCK-8 assay. Cell secretion inflammatory factor interleukin-23 (IL-23) was detected by ELISA. The expressions of intracellular chemokines CXCL1, CXCL2, CCL20 and 6-defensin 4 (DEFB4) were detected by Real-time PCR.. Shikonin (2,1 mg x L(-1)) could distinctly inhibit HaCaT cell proliferation induced by IL-17A, with statistical difference (P < 0.01). Each shikonin group showed decreases in the secretion of IL-23 and inhibition in expressions of intracellular CXCL1, CXCL2, CCL20 and DEFB4.. Shikonin could inhibit HaCaT cells proliferation induced by IL-17 and secretion of relevant cytokines and recruit leukocytes by inhibiting chemokines, so as to show the effect in treating psoriasis.

Topics: Cell Line; Cell Proliferation; Chemokines; Drugs, Chinese Herbal; Humans; Interleukin-17; Keratinocytes; Naphthoquinones

Osteosarcoma (OS) is the most common primary malignant bone tumor, notorious for its metastasis. We have recently shown that shikonin, an effective constituent extracted from Chinese medicinal herb, induces necroptosis in OS cells. Nevertheless, the effects of low-dose shikonin on the invasiveness of OS cells are unknown. Here, we showed that shikonin dose-dependently decreased OS cell invasiveness in both scratch wound healing assay and transwell cell migration assay. Moreover, the direct target of shikonin on cell invasiveness was found to be matrix metalloproteinase (MMP)-13. Further, the inhibitory effects of shikonin on cell invasiveness were completely abolished in MMP13-overexpressing OS cells. Together, these data suggest that shikonin may suppress OS invasiveness through MMP13 suppression. Thus, our data highlight a previous unappreciated role for shikonin in suppressing OS cell metastasis.

Topics: Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 13; Naphthoquinones; Neoplasm Invasiveness; Osteosarcoma

Shikonin, a natural naphthoquinone isolated from the Chinese traditional medicine Zi Cao (purple gromwell), is known to suppress the growth of several cancer cell types. In this study, we evaluated the pro-apoptotic effects of shikonin on MCF-7 and HeLa cells, and investigated the underlying mechanism. Shikonin-induced apoptosis was associated with activation of caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, up-regulation of p73, and down-regulation of BCL-2. Shikonin also induced up-regulation of the tumor suppressor gene, p16(INK4A). Increasing transcriptional activity of p16(INK4A) by shikonin treatment, we observed in luciferase promoter assay, reflects reduced promoter binding by down-regulation of ICBP90 (inverted CCAAT box binding protein, 90 kDa), which are involved in down-regulation of its partner, DNMT1 (DNA methyltransferase 1). On the basis of these results, we conclude that shikonin causes apoptosis via a p73-related, caspase-3-dependent pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; CCAAT-Enhancer-Binding Proteins; Cyclin-Dependent Kinase Inhibitor p16; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; HeLa Cells; Humans; Luciferases; MCF-7 Cells; Naphthoquinones; Nuclear Proteins; Poly(ADP-ribose) Polymerases; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Protein p73; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

Hypertrophic scarring/hypertrophic scars (HS) is a highly prevalent condition following burns and trauma wounds. Numerous studies have demonstrated that transforming growth factor-β1 (TGF‑β1) plays an essential role in the wound healing process by regulating cell differentiation, collagen production and extracellular matrix degradation. The increased expression of TGF-β1 is believed to result in the formation of HS. Shikonin (SHI), an active component extracted from the Chinese herb, Radix Arnebiae, has previously been found to downregulate the expression of TGF-β1 in keratinocyte/fibroblast co-culture conditioned medium. In view of this, in this study, we aimed to further investigate the effects of SHI on TGF-β1-stimulated hypertrophic scar-derived human skin fibroblasts (HSFs) and examined the underlying mechanisms. Cell viability and proliferation were measured using alamarBlue and CyQUANT assays. The total amount of collagen and cell contraction were examined using Sirius red staining and the cell contraction assay kit. Gene expression and signalling pathway activation were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Our results revealed that SHI reduced TGF-β1‑induced collagen production through the ERK/Smad signalling pathway and attenuated TGF-β1‑induced cell contraction by downregulating α-smooth muscle actin (αSMA) expression in the HSFs. The data from this study provide evidence supporting the potential use of SHI as a novel treatment for HS.

Topics: Actins; Cicatrix, Hypertrophic; Collagen; Down-Regulation; Fibroblasts; Humans; MAP Kinase Signaling System; Naphthoquinones; Skin; Transforming Growth Factor beta1

Shikonin is a major active chemical component extracted from Lithospermi Radix, an effective traditional herb in various types of wound healing. Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue. The purpose of the study was to investigate its mechanism of action in human skin cells.. MTS assay was used to measure cell growth. The collagen type I (COL1 ) mRNA expression and procollagen type I C-peptide (PIP) production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB (NF-κB) signaling pathway. Cell-based proteasome activity assay was used to determine proteasome activity.. In this study, we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts. However, shikonin did not directly induce COL1 mRNA expression and PIP production in dermal fibroblasts in vitro. In addition, 1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts. Furthermore, shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation of phosphorylated inhibitor κB-α in dermal fibroblasts.. These results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome. Thus, shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.

Topics: Cell Proliferation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Humans; Keratinocytes; Naphthoquinones; NF-kappa B; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Skin

Hypertrophic scarring is a highly prevalent condition clinically and results from a decreased number of apoptotic fibroblasts and over-abundant production of collagen during scar formation following wound healing. Our previous studies indicated that Shikonin, an active component extracted from Radix Arnebiae, induces apoptosis and reduces collagen production in hypertrophic scar-derived fibroblasts. In the study reported here, we further evaluate the potential use of Shikonin as a novel scar remediation therapy by examining the effects of Shikonin on both keratinocytes and fibroblasts using Transwell® co-culture techniques. The underlying mechanisms were also revealed. In addition, effects of Shikonin on the expression of cytokines in Transwell co-culture "conditioned" medium were investigated.. Our results indicate that Shikonin preferentially inhibits cell proliferation and induces apoptosis in fibroblasts without affecting keratinocyte function. In addition, we found that the proliferation-inhibiting and apoptosis-inducing abilities of SHI might be triggered via MAPK and Bcl-2/Caspase 3 signalling pathways. Furthermore, SHI has been found to attenuate the expression of TGF-β1 in Transwell co-cultured "conditioned" medium.. The data generated from this study provides further evidence that supports the potential use of Shikonin as a novel scar remediation therapy.

Topics: Apoptosis; Cell Line; Cicatrix, Hypertrophic; Coculture Techniques; Collagen; Fibroblasts; Humans; Keratinocytes; MAP Kinase Signaling System; Naphthoquinones

Though previous study demonstrated that shikonin could exert its antitumor activity by inducing apoptosis and necrosis, the pro-survival mechanisms involved in its antitumor process are still little to know. In the present study, for the first time, we found a protective mechanism was simultaneously activated which caused the reduced sensitivity of glioblastoma stem cells (GSCs) to the cytotoxicity of shikonin. Reduced active caspase-9 expression and enhanced mitochondrial membrane potential (MMP) were intriguingly observed within 24 h treatment by shikonin in GSCs. Further investigation identified that Endoplasmic Reticulum Stress (ERS) was involved in its antitumor process, which compromised the cytotoxicity of shikonin toward GSCs. Inhibiting ERS by 4-phenylbutyric acid (4-PBA) markedly enhanced the cytotoxicity of shikonin in GSCs. The consistent result was simultaneously observed in the GSCs-xenografted mice. Furthermore, our results identified that JNK/c-Jun pathway was involved in the antitumor process of shikonin, providing a mechanism by which ERS reduced the cytotoxicity of shikonin toward GSCs. Altogether, the novel observation in the present study identified that inhibiting ERS would be an attractive new approach to enhance the therapeutic potency of shikonin toward GSCs.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cells, Cultured; Drugs, Chinese Herbal; Endoplasmic Reticulum Stress; Glioblastoma; Humans; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Mice, Nude; Naphthoquinones; Neoplastic Stem Cells

Obesity represents a major public health problem, and identifying natural compounds that modulate energy balance and glucose homeostasis is of interest for combating obesity and its associated disorders. The naphthoquinone shikonin has diverse beneficial properties including anti-inflammatory, anti-oxidant, and anti-microbial effects. The objective of this study is to investigate the effects of shikonin on adiposity and glucose homeostasis.. The metabolic effects of shikonin treatment on mice fed regular chow or challenged with a high-fat diet (HFD) were determined.. Shikonin treated mice fed regular chow exhibited improved glucose tolerance compared with controls. In addition, shikonin treated mice fed HFD displayed decreased weight gain and resistance to HFD-induced glucose intolerance. Further, shikonin treatment decreased HFD-induced hepatic dyslipidemia. These findings correlated with enhanced hepatic insulin signaling in shikonin treated mice as evidenced by increased tyrosyl phosphorylation of the insulin receptor and enhanced downstream signaling.. These studies identify shikonin as a potential regulator of systemic glucose tolerance, energy balance, and adiposity in vivo.

Topics: Adiposity; Animals; Diet, High-Fat; Down-Regulation; Drugs, Chinese Herbal; Energy Metabolism; Glucose; Glucose Intolerance; Glucose Tolerance Test; Homeostasis; Insulin; Insulin Resistance; Liver; Male; Mice; Mice, Inbred C57BL; Naphthoquinones; Obesity; Weight Gain

Shikonin, a natural product from Lithospermum erythrorhizon, exerts a wide range of anti-inflammatory actions both in vitro and in vivo. Matrix metalloproteinases (MMPs) have long been considered as the major catabolic enzymes involved in osteoarthritis (OA) cartilage erosion. Here, we investigated the anti-inflammatory and effects of shikonin on MMPs in both IL-1β induced rabbit chondrocytes and the experimental rabbit OA model induced by anterior cruciate ligament (ACL) transection and evaluated the potential involvement of nuclear factor kappa B (NF-κB) in the processes. In vitro, rabbit chondrocytes were cultured and pretreated with shikonin (0, 1, 5, 10μM) for 1h (h) with or without IL-1β (10ng/ml) for 24h. The expression of MMPs (MMP-1, MMP-3 and MMP-13) and tissue inhibitors of metalloproteinase-1 (TIMP-1) at mRNA and protein levels were determined by quantitative real-time PCR and ELISA respectively. NF-κB related signaling molecules were investigated by Western blotting. In vivo study, the effects of shikonin on MMPs and TIMP-1 were determined at the gene level and the cartilage damage was evaluated at the histological level after the rabbits sacrificed. We found that shikonin significantly reversed the elevated expression of MMP-1, MMP-3 and MMP-13 and the reduced expression of TIMP-1 at both gene and protein levels in IL-1β induced chondrocytes. Additionally, the reduction of IκBα and the activation of NF-κB p65 induced by IL-1β were subsided by shikonin in rabbit chondrocytes. In vivo, both the cartilage damage and the elevated expression of MMP-1, MMP-3 and MMP-13 and the decreased expression of TIMP-1 were ameliorated in shikonin intra-articular injection knees compared to vehicle knees. Our findings indicated that shikonin have anti-inflammatory and chondro-protective effects and may be a potential therapeutic agent for the treatment of OA.

Topics: Animals; Cartilage; Cell Survival; Chondrocytes; Gene Expression Regulation; I-kappa B Kinase; Inflammation; Interleukin-1beta; Male; Matrix Metalloproteinases; Naphthoquinones; NF-kappa B; Osteoarthritis; Rabbits; Tissue Inhibitor of Metalloproteinase-1

The tumor cell lysate-pulsed, dendritic cell (DC)-based cancer vaccine approaches are being actively evaluated for application to cancer immunotherapy, hopefully at a personalized medicine base. There is apparently an emerging technical problem however, the lack of highly efficacious potency in activation of patient's DCs for T-cell priming and the associated process for presenting tumor immunogenicity.. One strategy to address this is to consider the manipulation of the tumor immunogenic cells death (ICD) complex ex-vivo for maximal activation of DC efficacy. In our previous study we showed that phytochemical shikonin (SK) can drastically enhance ICD activity in mouse tumor cells treated ex-vivo, and the resultant tumor cell lysate (TCL) can effectively augment such SK-TCL pulsed DC vaccine activity in vivo in anti-tumor activities. In this study, we investigated the specifics and the multi-functional effects of various damaged associated molecular pattern (DAMP) components of the ICD complex for their participation, roles and potential cross talks in activating DCs, as measured by five different functional assays.. Among three DAMPs tested, HSP70 and CRT mediate a key role in SK-TCL-induced DC immunity for both CD4(+) and CD8(+) T cell proliferations in vitro. HSP70 is the most important component, followed by CRT, then HMGB1 in facilitating DC immunity on suppressing metastasis of mouse 4 T1 mammary tumors and prolonging survival in test mice. Only HSP70, but not CRT or HMGB1, is effective for the suppression of both granulocytic and monocytic MDSC populations in vivo. Both HSP70 and HMGB1, but not CRT, are essential in activating the expression of three key ICD molecules-associated receptors on test DCs. Each of the three test ICD proteins can exhibit a distinguishable pattern in stimulating the expression of four key chemokines in test DCs.. Our findings on the differential roles or effect of various ICD components in activating vaccinated DCs may help formulate new strategies for future cancer vaccine designs.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Cell Proliferation; Dendritic Cells; HMGB1 Protein; HSP70 Heat-Shock Proteins; Humans; Immunotherapy; Lymphocyte Activation; Melanoma, Experimental; Mice; Naphthoquinones; Precision Medicine; T-Lymphocytes

Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan's National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine.

Topics: Adult; Animals; Autophagy; Cancer Vaccines; Cell Differentiation; Cell Line, Tumor; Dendritic Cells; Female; Humans; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Middle Aged; Naphthoquinones; Neoplasm Metastasis; Neoplasms; Sirolimus; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Young Adult

The aim of this study was to evaluate the antigenotoxic and antioxidant potential of shikonin (SH), acetylshikonin (ACS) and Arnebia euchroma callus extract (EXT). The antigenotoxic activity was investigated by the umu-test as the inhibition of the SOS system induction caused by genotoxic chemical agents - 4-nitroquinoline oxide and 2-aminoanthracene. Moreover the ability of SH, ACS and EXT to prevent photogenotoxicity triggered by chlorpromazine under UVA irradiation was measured. The cytotoxicity of EXT toward V79 Chinese hamster cell line was additionally assessed. Shikonin and acetylshikonin had no effect on 4-NQO induced genotoxicity whereas EXT demonstrated an unclear effect. The protection against 2AA induced genotoxicity was observed for all tested substances. The highest protection was demonstrated for EXT with inhibition of 66%. SH and ACS reduced 2AA genotoxicity with inhibition of about 60%. Under UVA the strongest and dose-dependent activity was observed for EXT. Acetylshikonin was a weak anti-photogenotoxin whereas shikonin had no clear effect. EXT was highly cytotoxic toward the V79 cell line - the cells' morphology was affected seriously and apoptosis was impacted. The antioxidant activity of SH, ACS and EXT was studied by means of electron paramagnetic resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. All three samples exhibited radical scavenging properties.

Topics: 4-Nitroquinoline-1-oxide; Animals; Anthracenes; Anthraquinones; Antioxidants; Boraginaceae; Cell Line; Chlorpromazine; Cricetinae; Cricetulus; Electron Spin Resonance Spectroscopy; Male; Mutagenicity Tests; Naphthoquinones; Plant Extracts; Rats; Rats, Sprague-Dawley

We hypothesized that interferon-γ (IFN-γ) induces K17 over-expression in HaCaT cells by activating STAT3 and that Sh might inhibit the over-expression through interference of STAT3 signaling.. In vitro culture of HaCaT cells treated with IFN-γ and measurement of K17 protein by enzyme linked immunosorbent assay.. The level of K17 protein (one kind of keratin protein) in the supernatant induced by IFN-γ was significantly reduced by Shikonin at various concentrations. Interference of STAT3 suppressed the effect of IFN-γ on K17 expression at both mRNA and protein levels. The over-expression of K17 in IFN-γ-induced HaCaT cells was significantly suppressed by 2 µg/L Shikonin. Interfering with STAT3 signaling with 2 µg/L Shikonin resulted in an intermediate level of IFN-γ-induced K17 protein in HaCaT cells.. These data demonstrate that IFN-γ induces K17 protein over-expression of HaCaT cells by activating STAT3 and Shikonin may inhibit the over-expression partly through interference of STAT3.

Topics: Cell Line; Humans; Interferon-gamma; Keratin-17; Keratinocytes; Naphthoquinones; Signal Transduction; STAT3 Transcription Factor; Up-Regulation

In this work, electrogenerated anion and dianion species from shikonin and its ester derivative isovalerylshikonin were characterized by means of ESR/UV-Vis spectroelectrochemistry. Analysis of the spectra supported the proposal that stepwise dissociative electron transfer (DET) takes place during the second reduction process of the esterified compound. Quantum chemical calculations were performed for validating the occurrence of this mechanistic pathway and for obtaining thermodynamic information on the electron transfer process; ΔG(cleavage)(0) was estimated to be -0.45 eV, considering that the two possible products of the overall reaction scheme are both a quinone and carboxylate anions.

Topics: Electrochemical Techniques; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Esters; Naphthoquinones; Pentanoic Acids; Quantum Theory; Spectrophotometry, Ultraviolet; Thermodynamics

Shikonin is a naturally occurring naphthoquinone pigment and a major constituent present in Lithospermum erythrorhizon. Since microRNAs (miRNAs) are one of the key post-transcriptional regulators of adipogenesis, their manipulation represents a potential new strategy to inhibit adipogenesis. Our aim was to investigate shikonin-dependent inhibition of adipogenesis with an emphasis on miRNA-related processes. Mir-34a increased during induced adipogenesis, and this was suppressed in the presence of shikonin. mRNA expression of FKBP1B, a suggested target of mir-34a according to bioinformatics studies, decreased during adipogenesis, but was recovered by shikonin treatment, which reversely correlated with mir-34a expression. A mir-34a inhibitor suppressed MDI-induced adipogenesis by blocking PPARγ and C/EBPα expression, while suppression of mir-34a recovered MDI-induced down-regulation of FKBP1B expression. A mir-34a mimic decreased FKBP1B mRNA expression in 3T3-L1 preadipocytes. We also observed that mir-34a bound directly to the 3'-untranslated region of FKBP1B. Finally, FKBP1B overexpression attenuated MDI-induced adipogenesis, PPARγ, and C/EBPα expression. These results suggest that mir-34a regulates adipogenesis by targeting FKBP1B expression. Our findings reveal that shikonin prevents adipogenesis by blocking the mir-34a-FKBP1B pathway which represents a promising potential target for preventing obesity.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Animals; Cell Differentiation; Mice; MicroRNAs; Naphthoquinones; Tacrolimus Binding Proteins

Leukemia remains life-threatening despite remarkable advances in chemotherapy. The poor prognosis and drug resistance are challenging treatment. Novel drugs are urgently needed. Shikonin, a natural naphthoquinone, has been previously shown by us to be particularly effective towards various leukemia cell lines compared to solid tumors. However, the underlying mechanisms are still poorly understood. Here, we investigated shikonin and 14 derivatives on U937 leukemia cells. Four derivatives (isobutyrylshikonin, 2-methylbutyrylshikonin, isovalerylshikonin and β,β-dimethylacrylshikonin) were more active than shikonin. AnnexinV-PI analysis revealed that shikonins induced apoptosis. Cell cycle G1/S check point regulation and the transcription factor c-MYC, which plays a vital role in cell cycle regulation and proliferation, were identified as the most commonly down-regulated mechanisms upon treatment with shikonins in mRNA microarray hybridizations. Western blotting and DNA-binding assays confirmed the inhibition of c-MYC expression and transcriptional activity by shikonins. Reduction of c-MYC expression was closely associated with deregulated ERK, JNK MAPK and AKT activity, indicating their involvement in shikonin-triggered c-MYC inactivation. Molecular docking studies revealed that shikonin and its derivatives bind to the same DNA-binding domain of c-MYC as the known c-MYC inhibitors 10058-F4 and 10074-G5. This finding indicates that shikonins bind to c-MYC. The effect of shikonin on U937 cells was confirmed in other leukemia cell lines (Jurkat, Molt4, CCRF-CEM, and multidrug-resistant CEM/ADR5000), where shikonin also inhibited c-MYC expression and influenced phosphorylation of AKT, ERK1/2, and SAPK/JNK. In summary, inhibition of c-MYC and related pathways represents a novel mechanism of shikonin and its derivatives to explain their anti-leukemic activity.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Naphthoquinones; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Signal Transduction; U937 Cells

Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drugs, Chinese Herbal; Glioblastoma; Humans; Insulin-Like Growth Factor I; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Medicine, Chinese Traditional; Naphthoquinones; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction

Shikonin (SK), a natural naphthoquinone isolated from the Chinese medicinal herb, has been known to suppress the proliferation of several cancer cells. However, its role in the epithelial mesenchymal transition (EMT) has yet to be demonstrated. The aim of the present study was to examine the effects of SK on EMT. Lipopolysaccharide (LPS) induced EMT-like phenotypic changes, enhancing cell migration and invasion. SK markedly reduced the expression of the LPS-induced EMT markers, including N-cadherin in MDA-MB‑231 cells, and increased the expression of E-cadherin in MCF-7 cells. SK also inhibited cell migration and invasion in vitro. The effects of SK on the LPS-induced EMT were mediated by the inactivation of the NF-κB-Snail signaling pathway. The results provided new evidence that SK suppresses breast cancer cell invasion and migration by inhibiting the EMT. Therefore, SK is a potentially effective anticancer agent for breast tumors, by inhibiting metastasis.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Female; Humans; Lipopolysaccharides; MCF-7 Cells; Microscopy, Fluorescence; Naphthoquinones; Snail Family Transcription Factors; Transcription Factor RelA; Transcription Factors

Breast cancer, the most common cancer in the women, is the leading cause of death. Necrotic signaling pathways will enable targeted therapeutic agents to eliminate apoptosis-resistant cancer cells. In the present study, the effect of shikonin on the induction of cell necroptosis or apoptosis was evaluated using the T-47D breast cancer cell line. The cell death modes, caspase-3 and 8 activities and the levels of reactive oxygen species (ROS) were assessed. Cell death mainly occurred through necroptosis. In the presence of Nec-1, caspase-3 mediated apoptosis was apparent in the shikonin treated cells. Shikonin stimulates ROS generation in the mitochondria of T-47D cells, which causes necroptosis or apoptosis. Induction of necroptosis, as a backup-programmed cell death pathway via ROS stimulation, offers a new strategy for the treatment of breast cancer.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Breast Neoplasms; Caspase 3; Cell Proliferation; Female; Humans; Mitochondria; Naphthoquinones; Necrosis; Reactive Oxygen Species; Tumor Cells, Cultured

Shikonin, shikonofuran and their derivatives are the main bioactive components of Zicao, a traditional Chinese herbal medicine prepared with the dried roots of Lithospermum erythrorhizon, Arnebia euchroma or Arnebia guttata. However, approaches on the systematic discovery and identification of shikonins and shikonofurans, especially unknown ones, are still not available. To address this issue, the gas-phase CID-fragmentation routes for the shikonins and shikonofurans were established by using ESI-QTOF-MS in the negative ion mode and low-energy collision induced dissociation tandem mass spectrometry (CID-MS/MS) in this study using seventeen standards. As a result, diagnostic product ions for rapid discovery and classification of shikonins and shikonofurans were determined. In addition, various mobile phase compositions and UHPLC elution programs were evaluated to achieve optimal separation efficiency and detection response of these types of analytes. Based on these findings, an integral approach using UHPLC-ESI-QTOF-MS and CID-MS/MS analyses together with a novel two steps data mining strategy was developed for systematic analysis of shikonins and shikonofurans in complex samples. Consequently, 58 compounds including 32 novel ones were efficiently discovered and identified from the crude extract of Zicao. Moreover, comparative analyses of the 58 chemical components in three Zicao species including Lithospermum erythrorhizon, Arnebia euchroma and Arnebia guttata samples were conducted using the established analytical method, which can be instructive for future utilization of Zicao and its related medicinal products.

Topics: Chromatography, High Pressure Liquid; Data Mining; Drugs, Chinese Herbal; Furans; Naphthoquinones; Tandem Mass Spectrometry

Osteosarcoma (OS) is a primary malignant bone tumor in humans, and is notorious mainly for its distal metastases. We have recently shown that Shikonin, an effective constituent extracted from Chinese medicinal herb, inhibits OS cell invasion through suppression of matrix metalloproteinase 13 (MMP13). However, the underlying mechanisms remain unknown.. Here, we studied the levels of tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2) in OS cells upon Shikonin treatment. TIPE2 levels were adapted in OS cell lines through transfection with plasmids carrying transgene or short-hairpin interference RNA (shRNA), and the effects of TIPE2 adaptation on MMP13 and cell invasiveness were evaluated by RT-qPCR, Western blot, ELISA and transwell cell migration assay, respectively. TIPE2 levels in OS specimens from patients were examined and correlated with cancer metastases and patient survival.. We found that Shikonin dose-dependently decreased MMP13 levels, and increased TIPE2 levels in two OS cell lines, U2OS and SaOS-2. Overexpression of TIPE2 in U2OS significantly suppressed MMP13 levels and cell invasiveness. Depletion of TIPE2 in SaOS-2 cells significantly increased MMP13 levels and cell invasiveness. Moreover, TIPE2 levels in OS specimens were significantly decreased, compared to adjacent non-cancer bone tissue. Lower TIPE2 levels correlated with higher incidence of metastases and worse 5-year survival.. TIPE2 mediates the suppressive effects of Shikonin on MMP13 in osteosarcoma cells, and TIPE2 may be a novel therapeutic target for OS.

Topics: Adolescent; Cell Line, Tumor; Child; Female; Humans; Intracellular Signaling Peptides and Proteins; Matrix Metalloproteinase 13; Naphthoquinones; Osteosarcoma

A method for simultaneous determination of the shikonin, acetyl shikonin and β, β'-dimethylpropene shikonin in Onosma hookeri and the chromatographic fingerprint was estabished by HPLC-DAD on an Agilent Zorbax SB-column with a gradient elution of acetonitrile and water at 0.8 mL x min(-1), 30 degrees C. The quality assessment was conducted by comparing the content difference of three naphthoquinone constituents, in combination with chromatographic fingerprint analysis and systems cluster analysis among 7 batches of radix O. hookeri. The content of the three naphthoquinone constituents showed wide variations in 7 bathces. The similarity value of the fingerprints of sample 5, 6 and 7 was above 0.99, sample 2 and 3 above 0.97, sample 3 and 4 above 0.90, and other samples larger than 0.8, which was in concert with the content of three naphthoquinone constituents. The 7 samples were roughly divided into 4 categories. The results above indicated that the using of this medicine is complex and rather spotty. The established HPLC fingerprints and the quantitative analysis method can be used efficiently for quality assessment of O. hookeri.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Naphthoquinones; Plant Roots

This study aims to explore the apoptotic function of shikonin on the papillary thyroid cancer cells and the related mechanism. The papillary thyroid cancer cell lines K1 and W3 and thyroid follicular epithelial cells NTHY-ORI 3-1 were treated with different concentrations of shikonin. Cell proliferation was tested. Morphological changes of the apoptotic cells were observed by Hoechst 33342 staining. The apoptosis rate of the papillary thyroid cancer cells was measured with flow cytometry. Changes of the cell cycle were explored. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Bcl-2 family proteins and caspase-3 expression with shikonin treatment was analyzed by real-time fluorescence polymerase chain reaction (PCR). Cell proliferation of K1 and W3 was inhibited by shikonin, and the inhibition was dose-time dependent. Papillary thyroid carcinoma cells treated by shikonin had no obvious cell cycle arrest but were observed with the higher apoptosis rate and the typical apoptotic morphological changes of the cell nucleus. JC-1 staining showed that shikonin reduced the mitochondrial membrane potential of papillary thyroid carcinoma cells. Real-time PCR results showed that shikonin significantly increased Bax and caspase-3 expression and upregulated Bcl-2 expression in a dose-dependent manner in papillary thyroid carcinoma cells. However, the NTHY-ORI 3-1 was almost not affected by shikonin treatment. Shikonin can inhibit K1 and W3 cell proliferation in a dose- and time-dependent manner, enhance Bax levels, reduce anti-apoptotic protein Bcl-2 levels, result in decreasing mitochondrial membrane potential and activating caspase-3 enzyme, and finally lead to apoptosis.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Naphthoquinones; Real-Time Polymerase Chain Reaction; Thyroid Cancer, Papillary; Thyroid Neoplasms

A series of shikonin derivatives (1-13) that were acylated selectively by various thiophene or indol carboxylic acids at the side chain of shikonin were synthesized, and their biological activities were also evaluated as potential tubulin inhibitors. Among them, compound 3 ((R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-(1H-indol-3-yl)propanoate) and compound 8 ((R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 2-(thiophen-3-yl)acetate) exhibited good antiproliferative activity of A875 (IC50  = 0.005 ± 0.001 μm, 0.009  ± 0.002 μm) and HeLa (IC50  = 11.84 ± 0.64 μm, 4.62  ± 0.31 μm) cancer cell lines in vitro, respectively. Shikonin (IC50  = 0.46 ± 0.002 μm, 4.80 ± 0.48 μm) and colchicine (IC50  = 0.75 ± 0.05 μm, 17.79 ± 0.76 μm) were used as references. Meanwhile, they also showed the most potent growth inhibitory activity against tubulin (IC50 of 3.96  ± 0.13 μm and 3.05 ± 0.30 μm, respectively), which were compared with shikonin (IC50  =  15.20 ± 0.25 μm) and colchicine (IC50  = 3.50 ± 0.35 μm). Furthermore, from the results of flow cytometer, we found compound 3 can really inhibit HeLa cell proliferation and has low cell toxicity. Based on the preliminary results, compound 3 with potent inhibitory activity in tumor growth may be a potential anticancer agent.

Topics: Antineoplastic Agents; Binding Sites; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Colchicine; HeLa Cells; Humans; Indoles; Molecular Docking Simulation; Naphthoquinones; Protein Structure, Tertiary; Thiophenes; Tubulin; Tubulin Modulators

Inflammatory damage plays an important role in cerebral ischemic pathogenesis and represents a new target for treatment of stroke. Shikonin has gained attention for its prominent anti-inflammatory property, but up to now little is known about shikonin treatment in acute ischemic stroke. The aim of this study was to evaluate the potential neuroprotective role of shikonin in cerebral ischemic injury, and investigate whether shikonin modulated inflammatory responses after stroke. Focal cerebral ischemia in male ICR mice was induced by transient middle cerebral artery occlusion. Shikonin (10 and 25 mg/kg) was administered by gavage once a day for 3 days before surgery and another dosage after operation. Neurological deficit, infarct volume, brain edema, blood-brain barrier (BBB) dysfunction, and inflammatory mediators were evaluated at 24 and 72 h after stroke. Compared with vehicle group, 25 mg/kg shikonin significantly improved neurological deficit, decreased infarct volume and edema both at 24 and 72 h after transient ischemic stroke, our data also showed that shikonin inhibited the pro-inflammatory mediators, including TLR4, TNF-α, NF-κB, and phosphorylation of p38MAPK in ischemic cortex. In addition, shikonin effectively alleviated brain leakage of Evans blue, up-regulated claudin-5 expression, and inhibited the over-expressed MMP-9 in ischemic brain. These results suggested that shikonin effectively protected brain against ischemic damage by regulating inflammatory responses and ameliorating BBB permeability.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Blood-Brain Barrier; Brain Ischemia; Claudin-5; Down-Regulation; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred ICR; Naphthoquinones; Neuroprotective Agents; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Stroke; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; Up-Regulation

The naphthoquinone pigment, shikonin, is a natural product derived from Lithospermum erythrorhizon and an active component of a Chinese traditional herbal therapeutic. We identified shikonin as a candidate for shortening the circadian period using real-time reporter gene assays based on NIH3T3-derived stable reporter cells. Period length that became shortened in cells incubated with shikonin or etoposide reverted to that of control cells after continued incubation without these compounds. These findings indicated that shikonin and etoposide shorten the circadian period reversibly and through similar mechanisms. Topoisomerase II (Top2)-specific decatenation assays confirmed that shikonin, liker etoposide, is a Top2 inhibitor. Shikonin was incorporated into the nucleus and Top2 was located in the Bmal1 promoter, suggesting the relationship between Bmal1 transcription and Top2 inhibition. Top2a siRNA also shortened period length, suggesting that Top2 is involved in this process. Promoter assays showed that Top2a siRNA, etoposide and shikonin reduce Bmal1 promoter activity. These findings indicated that Top2 is involved in Bmal1 transcription and influences the circadian period, and that shikonin is a novel contributor to the control of period length in mammalian cells.

Topics: Animals; Antigens, Neoplasm; ARNTL Transcription Factors; Circadian Rhythm; DNA Topoisomerases, Type II; DNA-Binding Proteins; Drugs, Chinese Herbal; Gene Knockdown Techniques; Mice; Naphthoquinones; NIH 3T3 Cells; Poly-ADP-Ribose Binding Proteins; Transcription, Genetic

The optical antipodes alkannin/shikonin (A/S) and their esters are potent pharmaceutical substances found in the roots of 150 Boraginaceous species. This study estimated and compared total and free A/S content and A/S enantiomeric ratio in roots of 11 Alkanna species (A. corcyrensis, A. tinctoria, A. pindicola, A. orientalis, A. methanaea, A. calliensis, A. graeca, A. primuliflora, A. stribrnyi, A. sieberi and A. noneiformis) growing wild in various Greek regions, to compare with cultivated species. It also re-characterized the chirality of A/S commercial samples, since most of them were misnamed by the providers. Several Alkanna species were collected (groups 1 and 3) and botanically identified, whereas some Alkanna species were cultivated from collected seeds (group 2). Free A/S and derivatives were extracted from the dried roots of Alkanna species and analyzed by high performance liquid chromatography-diode array detection (HPLC-DAD). For total A/S content the hexane extracts of Alkanna roots were hydrolyzed and analyzed by HPLC-DAD. Chirality determination and A/S enantiomeric ratio estimation was performed for several commercial samples by polarimetry,chiral LC-DAD and circular dichroism studies. Quantitative analysis revealed that A/S content varied from one region to another even within the same species. Most of the cultivated samples contained greater amounts of free and total A/S compared with the wild ones, wheras no difference was observed in A/S enantiomeric ratio. All the Alkanna samples tested contain mainly alkannin derivatives. Some of the examined Alkanna species of the Greek flora that are endemic to the Mediterranean area could serve as alternative sources for medicinally valuable A/S derivatives. Most of the commercial A/S samples tested were misnamed in terms of chirality and re-characterized.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Naphthoquinones; Plant Extracts; Plant Roots

Lithospermum erythrorhizon, a naphthoquinone compound derived from a shikonin, has long been used as traditional Chinese medicine for treatment of various diseases, including cancer. To evaluate the cytotoxic effects of shikonin on AGS gastric cancer cells via induction of cell cycle arrest.. We observed the effects of 12.5-100 ng/mL dosage of shikonin treatment on AGS cancer cell line with the incubation time of 6h. Cytotoxic effects were assessed by measuring the changes in the intracellular ROS, appearance of senescence phenotype, cell cycle progression, CDK and cyclins expression levels upon shikonin treatment. We also examined upon the activation of Egr1-mediated p21 expression, by siRNA transfection, Luciferase assay, and ChIP assay.. In this study, we found that shikonin inhibits cell proliferation by arresting cell cycle progression at the G2/M phase via modulation of p21 in AGS cells. Also, our results revealed that the p21 gene was transactivated by early growth response1 (Egr1) in response to the shikonin treatment. Transient Egr1 expression enhanced shikonin-induced p21 promoter activity, whereas the suppression of Egr1 expression by small interfering RNA attenuated the ability of shikonin to induce p21 promoter activity.. Our results suggested that the anti-proliferative activity of shikonin was due to its ability to induce cell cycle arrest via Egr1-p21 signaling pathway. Thus, the work stated here validates the traditional use of shikonin in the treatment of cancer.

Topics: Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Humans; Naphthoquinones; RNA, Small Interfering; Stomach Neoplasms

Suspension cultures of Arnebia euchroma supported with liquid perfluorodecalin (PFD) degassed, aerated, or ethylene-saturated were investigated as a novel in situ extraction system for enhanced alkannin/shikonin production. Simultaneously, the effect of PFD applied as the liquid gas carrier on the growth of A. euchroma biomass was studied. The similar dry (4-fold) and fresh (7-fold) biomass increase was observed in the control (without PFD addition) and supplemented with PFD-degassed or PFD-aerated cultures while PFD-ethylene application impeded cell growth. The highest total of alkannin/shikonin production (23.23 mg flask(-1)) was observed when PFD-aerated has been used and it resulted in about 50% higher yield of alkannin/shikonin compared with the control culture. Chiral HPLC analysis revealed that in cultures supported with PFD, both alkannin and shikonin were produced. Their mutual ratio varied depending on culture conditions, and the accumulation of alkannin prevailed under almost all culture conditions. PFD has proved to be exceptionally efficient and cell-safe solvent for the in situ extraction of naphthoquinone red pigments without exerting any detrimental effects on cell growth. Extracellularly secreted red naphthoquinones were easily dissolved and extracted from the PFD phase, which can be regenerated and reused (e.g., in continuous culture system).

Topics: Biomass; Boraginaceae; Chromatography, High Pressure Liquid; Culture Media; Drugs, Chinese Herbal; Ethylenes; Fluorocarbons; Naphthoquinones; Plant Growth Regulators; Plant Somatic Embryogenesis Techniques

To study the anti-inflammation effect of Shikonin (Shik) and its mechanism, murine macrophage-like RAW264.7 cells (RAW264.7 cells) were divided into control group, LPS group (0.125, 0.25 and 0.5μg/ml), LPS (0.125, 0.25 and 0.5μg/ml) plus Shik (0.5, 1 and 2μM) group, and Shik (2μM) group. After exposure for 24h, the levels of Interleukin-6 (IL-6), nitric oxide (NO) and Tumor Necrosis Factor-α (TNF-α) in supernatant were measured with ELISA, the expression of high mobility group box 1(HMGB1) in supernatant and cytoplasm was assayed using qRT-PCR, western blot and immunofluorescence assays, the expression of IFN-β in cellular and supernatant was assayed by qRT-PCR and ELISA, and the ratio of nuclear to cytoplasm for NF-κB protein expression was assayed using western blot. The results of our investigation demonstrated that Shik could reduce significantly the levels of IL-6, NO and TNF-α in RAW264.7 cells exposed to LPS (P<0.05 or P<0.01). The expression of HMGB1, IFN-β and the ratio of nuclear to cytoplasm for NF-κB protein expression in LPS plus Shik group declined significantly as compared with LPS group (P<0.05 or P<0.01). The inhibitors of IFN-β signaling molecule JAK and NF-κB could attenuate significantly the expression of HMGB1 in supernatant. It was found in the present study that Shik could have the anti-inflammatory effects in RAW264.7 cells exposed to LPS, and one of the mechanisms may be the down-regulation of HMGB expression, which was associated with the IFN-β and NF-κB signaling pathways.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; HMGB1 Protein; Interferon-beta; Interleukin-6; Lipopolysaccharides; Mice; Naphthoquinones; Nitric Oxide; Transcription Factor RelA; Tumor Necrosis Factor-alpha

The effects of shikonin on gastric cancer cells were investigated in this study. Exposure to shikonin reduced the viability of gastric cancer cells in a time- and dose-dependent manner. However, apoptosis was not observed in gastric cancer cell treatment with different concentrations of shikonin for 6h. By contrast, treatment with shikonin for 24h significantly induced apoptosis, as evidenced by the results of TUNEL assay and flow cytometry analysis in proportion to the concentration. Disruption of the mitochondrial membrane potential was observed in gastric cancer cells that were treated with shikonin for 6 and 24h. Pretreatment with necrostatin-1 recovered cell death and mitochondrial membrane potential in the 6h shikonin treatment, but not in the 24h shikonin treatment. Western blot results reveal enhanced p38 phosphorylation, downregulated AKT phosphorylation, and increased caspase3 and PARP cleavage in cells that were treated with shikonin for 24h, but not in cells treated for 6h. Shikonin also triggered reactive oxygen species (ROS) generation both in the 6 and 24h treatments. Pretreatment with N-acetylcysteine blocked shikonin-induced cell death. In summary, our findings suggest that shikonin, which may function as a promising agent in the treatment of gastric cancers, sequentially triggered necrosis or apoptosis through ROS generation in gastric cancer cells.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Flow Cytometry; Free Radical Scavengers; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Necrosis; Phosphorylation; Reactive Oxygen Species; Stomach Neoplasms; Time Factors

IL-17 signaling in keratinocytes plays an important role in psoriasis, which is a benign, chronic skin disease characterized by keratinocytes hyperproliferation and increased dermal vascularity. Shikonin, isolated from the traditional medical herbs Lithospermum erythrorhizon, has long been found to possess different medicinal properties such as antibacterial, improving wound healing, anti-inflammatory and anti-tumor effects. However, the effects and mechanisms of shikonin on VEGF expression in keratinocytes mediated by IL-17 signaling, are still not fully clarified. In the present study, we investigated the effects and regulatory mechanisms of shikonin on VEGF expression in keratinocytes induced by IL-17 by in vitro and in vivo experiments. Our results showed that shikonin significantly inhibited IL-17-induced VEGF mRNA and protein expression in HaCaT cells and the secretion of VEGF by HaCaT cells, inhibited IL-17-induced IL-17R, pJAK2 and pSTAT3 expression, while up-regulated the expression of SOCS1 in HaCaT cells. Additionally, shikonin effectively suppressed VEGF expression in the skin of IL-17 stimulated mice. Furthermore, shikonin suppressed VEGF-induced tube formation of HUVECs and CD34 expression in the skin of IL-17 stimulated mice. These results imply that shikonin suppresses IL-17-induced VEGF expression in vitro and in vivo and the mechanisms may be related to its effect in blockage of JAK2/STAT3 pathway. These data deepen our understanding of shikonin in the inhibition of angiogenesis in inflammatory skin diseases such as psoriasis.

Topics: Angiogenesis Inhibitors; Animals; Cell Differentiation; Cell Line; Cell Survival; Cells, Cultured; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-17; Janus Kinase 2; Mice; Mice, Inbred BALB C; Naphthoquinones; STAT3 Transcription Factor; Vascular Endothelial Growth Factor A

Although gemcitabine is currently the best chemotherapeutic agent available for the treatment of advanced pancreatic cancer, eventual failure of response is a significant clinical problem. Therefore, novel therapeutic approaches against this disease are highly needed. The aim of this study was to evaluate whether shikonin, a naphthoquinone derivative, has potential in the treatment of pancreatic cancer when used either alone or in combination with gemcitabine. Our in vitro results showed that shikonin inhibited the proliferation of three different human pancreatic cancer cell lines and potentiated the cytotoxic effect of gemcitabine, which correlated with the down-regulation of constitutive as well as gemcitabine-induced activation of NF-κB and NF-κB-regulated gene products. Most importantly, using a xenograft model of human pancreatic cancer, we found shikonin alone significantly suppressed tumor growth and argumented the antitumor activity of gemcitabine. These effects also correlated with the down-regulation of NF-κB activity and its target genes, decreased proliferation (PCNA and Ki-67), decreased microvessel density (CD31), and increased apoptosis (TUNEL) in tumor remnants. Collectively, our results suggest that shikonin can suppress the growth of human pancreatic tumors and potentiate the antitumor effects of gemcitabine through the suppression of NF-κB and NF-κB-regulated gene products.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Survival; Deoxycytidine; Drug Synergism; Gemcitabine; Humans; Male; Mice; Mice, Nude; Microvessels; Naphthoquinones; Neovascularization, Pathologic; NF-kappa B; Pancreatic Neoplasms; Signal Transduction; Xenograft Model Antitumor Assays

Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been shown to possess tumor cell killing activity. The human androgen receptor (AR) is a nuclear transcription factor that serves as a major therapeutic target for prostate cancer. However, AR regulation by shikonin has not been reported. We investigated the effects of shikonin on the growth of prostate cancer cells. We observed that shikonin decreased the expression of AR at both the mRNA and the protein levels in LNCaP and 22RV1 human prostate cancer cells. The results from a luciferase assay showed that shikonin decreased the transcriptional activity of AR. Moreover, shikonin treatment inhibited AR target gene expression, PSA and growth inhibition of prostate cancer cells. In conclusion, the present study shows for the first time that shikonin treatment causes transcriptional repression of AR and inhibition of its nuclear localization in human prostate cancer cells. We propose that shikonin, an anticancer drug extracted from natural sources, induces inhibition of cell growth through modulation of AR in androgen-responsive prostate cancer cells and is a candidate for use in cancer chemotherapy for human prostate cancer.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Male; Naphthoquinones; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction

Shikonin, a major active component of the Chinese herbal plant Lithospermum erythrorhizon, has been applied for centuries in traditional Chinese medicine. Although shikonin demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of shikonin have not been fully defined. We report here that shikonin may interact with the cytosolic thioredoxin reductase (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme with a C-terminal -Gly-Cys-Sec-Gly active site, to induce reactive oxygen species (ROS)-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Shikonin primarily targets the Sec residue in TrxR1 to inhibit its physiological function, but further shifts the enzyme to an NADPH oxidase to generate superoxide anions, which leads to accumulation of ROS and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 attenuates the cytotoxicity of shikonin, whereas knockdown of TrxR1 sensitizes cells to shikonin treatment. Targeting TrxR1 with shikonin thus discloses a previously unrecognized mechanism underlying the biological activity of shikonin and provides an in-depth insight into the action of shikonin in the treatment of cancer.

Topics: Antioxidants; Apoptosis; Cell Survival; Drugs, Chinese Herbal; Gene Knockdown Techniques; HL-60 Cells; Humans; Leukemia; Naphthoquinones; Reactive Oxygen Species; Thioredoxin-Disulfide Reductase

The ability of pegylated liposomes (sterically stabilized liposomes-SSL) to localize in solid tumors via the enhanced permeability and retention (EPR) effect, partly depends on their long circulating properties which can be achieved by grafting polyethylene glycol (PEG) to the liposomes' surface. Alkannin and shikonin (A/S) are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant, and recently established antitumor activity. The purpose of this work was to prepare and characterize shikonin-loaded pegylated liposomes as a new drug carrier for shikonin, as a continuation of authors' previous work on conventional shikonin-loaded liposomal formulations. Three new pegylated liposomal formulations of shikonin (DSPC-PEG2000, EPC-PEG2000, and DPPC-PEG2000) were prepared and characterized in terms of physicochemical characteristics, pharmacokinetics, and stability (at 4 °C, for 28 d) and compared with the corresponding conventional ones. Particle size distribution, ζ-potential, entrapment efficiency, and release profile of the entrapped drug were measured. Results indicated the successful incorporation of shikonin into liposomes alongside with their good physicochemical characteristics, high entrapment efficiency, satisfactory in vitro release profile, and good physical stability. The results are considered promising and could be used as a road map for designing further in vivo experiments.

Topics: Antineoplastic Agents; Chemistry, Pharmaceutical; Drug Carriers; Drug Delivery Systems; Liposomes; Naphthoquinones; Particle Size; Phosphatidylethanolamines; Polyethylene Glycols

Cell suspension cultures of Arnebia euchroma were established from the friable callus on liquid Murashige and Skoog medium supplemented with 6-benzylaminopurine (10.0 μM) and indole-3-butyric acid (5.0 μM). Salicylic acid was used to study its effect on the enzymes which participate in shikonin biosynthesis with respect to metabolite (shikonin) content in the cell suspension culture of A. euchroma. In our study, phenylalanine ammonia lyase and PHB geranyltransferase were selected from the entire biosynthetic pathway. Results showed that phenylalanine ammonia lyase is responsible for growth and PHB geranyltransferase for metabolite production. Salicylic acid exhibited an inverse relationship with the metabolite content (shikonin); salicylic acid (100 μM) completely inhibited shikonin biosynthesis. The results presented in the current study can be successfully employed for the metabolic engineering of its biosynthetic pathway for the enhancement of shikonin, which will not only help in meeting its industrial demand but also lead to the conservation of species in its natural habitat.

Topics: Biosynthetic Pathways; Boraginaceae; Cell Culture Techniques; Culture Media; Geranyltranstransferase; Hydroxybenzoates; Naphthoquinones; Phenylalanine Ammonia-Lyase; Plant Proteins; Plants, Medicinal; Salicylic Acid

Shikonin, a small-molecule natural product which inhibits the activity of pyruvate kinase M2 (PKM2), has been studied as an anti-cancer drug candidate in human cancer models. Here, our results demonstrate that shikonin is able to sensitize human breast cancer cells to chemotherapy by paclitaxel (taxol). Human breast adenocarcinoma MBA-MD-231 cells, which have higher levels of PKM2 expression and activity compared with MCF-7 cells, were selected to study further. The concentrations of shikonin and taxol were first selected at which they did not significantly induce cytotoxicity when treated alone, whereas the combination induced apoptosis. Surprisingly, PKM2 activity was decreased by shikonin, but not by the combination treatment. To identify the potential targets of this combination, human phospho-kinase antibody array analysis was performed and results indicated that the combination treatment inhibited the activation of ERK, Akt, and p70S6 kinases, which are known to contribute to breast cancer progression. Finally, how the combination affects breast cancer cell growth in vivo was tested using a xenograft tumor model. The results indicated that shikonin plus taxol prolonged animal survival and reduced tumor size than the vehicle treatment group. In summary, our results suggest that shikonin has a potential as an adjuvant for breast cancer therapy.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Drug Interactions; Drug Therapy, Combination; Female; Heterografts; Humans; Mice; Mice, Nude; Naphthoquinones; Paclitaxel

Shikonin is a quinone-containing natural product that induces the apoptotic death of some cancer cell lines in culture through increasing intracellular reactive oxygen species (ROS). Quinone-based drugs have shown potential in the clinic, making shikonin an interesting compound to study. Our previous study found that shikonin induces apoptosis in neuroblastoma by induction of ROS, but its mechanism of action and scope of activity are unknown. In this study, we investigated the mode of oxidative stress of shikonin in human glioma cells. ROS induction by shikonin was of mitochondrial origin, as demonstrated by detection of superoxide with MitoSOX Red. Pre-incubation of shikonin with inhibitors of different complexes of the respiratory chain suggested that shikonin-induced ROS production occurred via complex II. In addition, NADPH oxidase and lipooxygenase are two other main ROS-generated sites in shikonin treatment. ROS production by shikonin resulted in the inhibition of nuclear translocation of Nrf2. Stable overexpression of Nrf2 in glioma cells inhibited ROS generation by shikonin. ROS generation from mitochondrial complex II, NADPH oxidase and lipooxygenase is likely the primary mechanism by which shikonin induces apoptosis in glioma cells. These findings also have relevance to the development of certain ROS producers as anti-cancer agents. These, along with shikonin have potential as novel chemotherapeutic agents on human glioma.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cytochromes c; Cytosol; Glioma; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Superoxides

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, has been reported to promote tumor cell death. However, there are few reports concerning its effect on metastasis-related cell invasion and migration behavior. In the present study, we investigated the effect of shikonin on human breast cancer invasion and migration. We found that shikonin inhibited phorbol 12-myristate 13-acetate (PMA)-induced cell migration and invasion in MCF-7 breast cancer cells, which was correlated with modulation of matrix metalloproteinase-9 (MMP-9) through suppression of both expression and proteolytic and promoter activity. We also found that shikonin inhibited both MMP-9 expression and promoter activity in MDA-MB‑231 cells with high metastatic potential. These results indicated that shikonin induces the suppression of migration and invasion through modulation of MMP-9 in human breast cancer cells. Therefore, shikonin may be a potential anticancer drug for human breast cancer therapy.

Topics: Breast Neoplasms; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; MCF-7 Cells; Naphthoquinones; Neoplasm Invasiveness; Signal Transduction; Tetradecanoylphorbol Acetate

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.

Topics: Antibodies, Monoclonal; Antigens, CD; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Diffusion; Drugs, Chinese Herbal; Female; Humans; Molecular Targeted Therapy; Nanocapsules; Nanocomposites; Naphthoquinones; Ovarian Neoplasms; Particle Size; Treatment Outcome

In this study, we report the identification of a new shikonin-phenoxyacetic acid derivative, as an inhibitor of tubulin. A series of compounds were prepared; among them, compound 16 [(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 2-(4- phenoxyphenyl) acetate] potently inhibited the function of microtubules, inducing cell growth inhibition, apoptosis of cancer cell lines in a concentration and time-dependent manner. Molecular docking involving 16 at the vinblastine binding site of tubulin indicated that a phenoxy moiety interacted with tubulin via hydrogen bonding with asparaginate (Asn) and tyrosine (Tyr). Analysis of microtubules with confocal microscopy demonstrated that 16 altered the microtubule architecture and exhibited a significant reduction in microtubule density. Cell cycle assay further proved that HepG2 cells were blocked in G2/M phase. Our study provides a new, promising compound for the development of tubulin inhibitors by proposing a new target for the anticancer activity of shikonin.

Topics: Antineoplastic Agents; Apoptosis; Binding Sites; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Hydrogen Bonding; Microtubules; Molecular Docking Simulation; Molecular Targeted Therapy; Naphthoquinones; Tubulin; Tubulin Modulators; Vinblastine

Shikonin, the most important component of Lithospermum erythrorhizon, has previously been shown to exert antioxidant, anti-inflammatory, antithrombotic, antiviral, antimicrobial and anticancer effects. The anticancer effect has been attributed to the stimulation of suicidal cell death or apoptosis. Similar to the apoptosis of nucleated cells, erythrocytes may experience eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include the increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide formation. The present study explored whether Shikonin stimulates eryptosis. To this end, Fluo 3 fluorescence was measured to quantify [Ca2+]i, forward scatter to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to determine hemolysis and antibodies to quantify ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Shikonin (1 µM) significantly increased [Ca2+]i, increased ceramide abundance, decreased forward scatter and increased annexin V binding. The effect of Shikonin (1 µM) on annexin V binding was significantly blunted, but not abolished by the removal of extracellular Ca2+. In conclusion, Shikonin stimulates suicidal erythrocyte death or eryptosis, an effect at least partially due to the stimulation of Ca2+ entry and ceramide formation.

Topics: Adenosine Triphosphate; Biological Transport; Calcium; Erythrocytes; Humans; In Vitro Techniques; Naphthoquinones; Phosphatidylserines

Shikonin, a naphthoquinone derived from the Chinese medicinal plant Lithospermum erythrorhizon, shows potential to be a cancer chemotherapeutic agent. Our previous data demonstrate that high doses (about 6 μM) of shikonin induce apoptosis in human hepatocellular carcinoma (HCC) cells. Here, we discovered that a low dose of shikonin (2.5 μM) and a short treatment time (12h) induced autophagy, as evidenced by the upregulation of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, the formation of acidic autophagic vacuoles (AVOs), and the punctate fluorescence pattern of GFP-LC3 protein. Next, we investigated the mechanism and found reactive oxygen species accumulation after shikonin treatment. The reactive oxygen species scavengers NAC and Tiron completely blocked autophagy. We further found activation of ERK by generation of reactive oxygen species and inhibition of RIP pathway, which are at least partially connected to shikonin-induced autophagy. Moreover, experiments in vivo revealed similar results: shikonin caused the accumulation of reactive oxygen species and phospho-ERK and thus induced autophagy in a tumor xenograft model. These findings suggest that shikonin is an inducer of autophagy and may be a promising clinical antitumor drug.

Topics: Animals; Antineoplastic Agents; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Intracellular Space; Liver Neoplasms; Male; Mice; Naphthoquinones; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Metastasis is one of the most important factors related to prostate cancer therapeutic efficacy. In previous studies, shikonin, an active naphthoquinone isolated from the Chinese medicine Zi Cao, has various anticancer activities both in vivo and in vitro. However, the mechanisms underlying shikonin's anticancer activity are not fully elucidated on prostate cancer cells. In the present study, we aimed to investigate the potential effects of shikonin on prostate cancer cells and the underlying mechanisms by which shikonin exerted its actions. With cell proliferation, flow cytometric cell cycle, migration and invasion assays, we found that shikonin potently suppressed PC-3 and DU145 cell growth by cell cycle arrest at the G2 phase and metastasis in a dose-dependent manner. Mechanically, we presented that shikonin could suppress the metastasis of PC-3 and DU145 cells via inhibiting the matrix metalloproteinase-2 (MMP-2) and MMP-9 expression and activation. In addition, shikonin significantly decreased the phosphorylation of AKT and mTOR in a dose-dependent manner while it induced extracellular signal-regulated kinase (ERK), p38 mitogen activated protein kinase (MAPK) and c-Jun N terminal kinase (JNK) phosphorylation. Further investigation of the underlying mechanism revealed that shikonin also induced the production of reactive oxygen species (ROS) that was reversed by the ROS scavenger dithiothreitol (DTT). Additionally, DTT reversed the shikonin induced activation of ERK1/2, thereby maintaining MMP-2 and MMP-9 expression and restoring cell metastasis. Together, shikonin inhibits aggressive prostate cancer cell migration and invasion by reducing MMP-2/-9 expression via AKT/mTOR and ROS/ERK1/2 pathways and presents a potential novel alternative agent for the treatment of human prostate cancer.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; G2 Phase; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthoquinones; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases

To explore the effects of Chinese herbal medicine radix rehmanniae, radix arnebiae and cortex moutan on the proliferation of HaCaT cells and explore their potential curative mechanisms.. The main monomers of catalpol, l-shikonin and paeonol were extracted. And 10 ng/ml keratinocyte growth factor (KGF) was used to induce HaCaT cell to build an in vitro model of hyperproliferation of epidermal keratinocytes. CCK-8 assay and flow cytometry were applied to examine the effects of herbal monomers on cell proliferation and cell cycle.. Both l-shikonin ( ≥ 10(-6) mol/L) and paeonol ( ≥ 1.88×10(-4) mol/L) inhibited cell proliferation while catalpol ( ≥ 10(-6) mol/L) enhanced cell proliferation.L-shikonin ( ≥ 10(-6) mol/L) and paeonol ( ≥ 1.88×10(-4) mol/L) inhibited the HaCaT cell during S and G2M phases while catalpol ( ≥ 10(-6) mol/L) enhanced HaCaT cell during S phase but not G2M phase.. L-shikonin and paeonol inhibits the proliferation of HaCaT cells while catalpol has opposite effects.

Topics: Acetophenones; Cell Line; Cell Proliferation; Humans; Iridoid Glucosides; Naphthoquinones

Shikonin is a compound from the herbal plant Lithospermum erythrorhizon that has been proved to possess powerful anti-proliferative effect on many kinds of cancers and to be safe in in vivo study. Posterior capsular opacification (PCO), the most frequent complication of cataract surgery, is mainly caused by the uncontrolled proliferation of retained human lens epithelial cells (HLEs). In this study, we investigated the effect of shikonin on the proliferation of HLEs and explored its underlying mechanism of action. Shikonin significantly inhibited the proliferation of HLEs in a dose- and time-dependent manner. Its anti-proliferative effect was exerted through induction of apoptosis. Reactive oxygen species (ROS) generation played an essential role in this apoptotic process. Interestingly, scavenging of ROS completely blocked the apoptosis induced by shikonin. In addition, the treatment of shikonin in HLEs significantly increased the ratio of Bax/Bcl-2, disrupted mitochondria membrane potential (MMP) and activated caspases. The inhibition of caspase largely blocks the apoptosis. The changes of MAPK pathway were also demonstrated. Shikonin effectively inhibited the phosphorylation of ERK, while it activated the phosphorylation of JNK and p38. These results suggested that shikonin inhibited the proliferation of HLEs by inducing apoptosis through ROS generation and the caspase-dependent pathway and the MAPK pathway was also involved.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 7; Caspase 9; Caspase Inhibitors; Cell Line; Cell Proliferation; Drugs, Chinese Herbal; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Lens, Crystalline; Membrane Potential, Mitochondrial; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, can exert inhibitory effect on tumor cell growth. However, little has been known concerning the effect of shikonin on lung adenocarcinoma cell and underlying mechanisms. In the present study, we investigated the effect of shikonin on the proliferation, cell cycle arrest, and apoptosis in human lung adenocarcinoma cells. We found that shikonin significantly suppressed the proliferation of lung adenocarcinoma cells compared with control in dose- and time-dependent manner (P < 0.05). In the meantime, our results showed that shikonin markedly increased the proportion of A549 cells at stage G1 as well as induced apoptosis in A549 cells. Furthermore, suppressed CCND1 and elevated caspase3 and caspase7 expression levels at mRNA were found in this study, indicating that shikonin may inhibit the growth of lung adenocarcinoma cell by changing cell cycle and promoting cell apoptosis through the regulation of CCND1, caspase3, and caspase7. Although more studies are needed, this study suggests that shikonin has the potential to be used as an anti-cancer agent in the treatment of lung adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Naphthoquinones; Resting Phase, Cell Cycle

The shikonin derivatives, accumulated in the roots of Arnebia euchroma (Boraginaceae), showed antibacterial, anti-inflammatory, and anti-tumor activities. To explore their possible biosynthesis regulation mechanism, this paper investigated the effects of exogenous methyl jasmonate (MJ) on the biosynthesis of shikonin derivatives in callus cultures of A. euchroma. The main results include: Under MJ treatment, the growth of A. euchroma callus cultures was not inhibited, but the expression level of both the genes involved in the biosynthesis of shikonin derivatives and their precursors and the genes responsible for intracellular localization of shikonin derivatives increased significantly in the Red Strain (shikonin derivatives high-producing strain). The quantitative analysis showed that six out of the seven naphthoquinone compounds under investigation increased their contents in the MJ-treated Red Strain, and in particular, the bioactive component acetylshikonin nearly doubled its content in the MJ-treated Red Strain. In addition, it was also observed that the metabolic profiling of naphthoquinone compounds changed significantly after MJ treatment, and the MJ-treated and MJ-untreated strains clearly formed distinct clusters in the score plot of PLS-DA. Our results provide some new insights into the regulation mechanism of the biosynthesis of shikonin derivatives and a possible way to increase the production of naphthoquinone compounds in A. euchroma callus cultures in the future.

Topics: Acetates; Boraginaceae; Cell Culture Techniques; Cyclopentanes; Naphthoquinones; Oxylipins; Plant Proteins; Plant Roots

An electrochemical and theoretical analysis of a series of shikonin derivatives in aprotic media is presented. Results showed that the first electrochemical reduction signal is a reversible monoelectronic transfer, generating a stable semiquinone intermediate; the corresponding E(I)⁰ values were correlated with calculated values of electroaccepting power (ω(+)) and adiabatic electron affinities (A(Ad)), obtained with BH and HLYP/6-311++G(2d,2p) and considering the solvent effect, revealing the influence of intramolecular hydrogen bonding and the substituting group at position C-2 in the experimental reduction potential. For the second reduction step, esterified compounds isobutyryl and isovalerylshikonin presented a coupled chemical reaction following dianion formation. Analysis of the variation of the dimensionless cathodic peak potential values (ξ(p)) as a function of the scan rate (v) functions and complementary experiments in benzonitrile suggested that this process follows a dissociative electron transfer, in which the rate of heterogeneous electron transfer is slow (~0.2 cm s(-1)), and the rate constant of the chemical process is at least 10(5) larger.

Topics: Electrochemical Techniques; Electron Transport; Esters; Molecular Structure; Naphthoquinones; Quantum Theory

A set of forty alkannin and shikonin oxime derivatives were firstly designed and synthesized. Their cytotoxicities against three kinds of tumor cells and a normal cell line were tested and compared with alkannin and shikonin. The cell-based investigation demonstrated that some oxime derivatives were more or comparatively effective to the lead compounds, especially their selective and excellent antitumor activities towards K562 cells with no toxicity in normal cells. We may conclude that oximate modification to the mother nucleus of alkannin and shikonin is an available approach to acquire potent antitumor agents.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; K562 Cells; MCF-7 Cells; Molecular Structure; Naphthoquinones; Oximes; Structure-Activity Relationship

Oxidative stress damages dermal and epidermal cells and degrades extracellular matrix proteins, such as collagen, ultimately leading to skin aging. The present study evaluated the potential protective effect of the aqueous methanolic extract obtained from Lithospermum erythrorhizon (LE) against oxidative stress, induced by H2O2 and ultraviolet (UV) irradiation, on human keratinocyte (HaCaT) and human dermal fibroblast-neonatal (HDF-n) cells. Exposure of cells to H2O2 or UVB irradiation markedly increased oxidative stress and reduced cell viability. However, pretreatment of cells with the LE extract not only increased cell viability (up to 84.5%), but also significantly decreased oxidative stress. Further, the LE extract downregulated the expression of matrix metalloproteinase-1, an endopeptidase that degrades extracellular matrix collagen. In contrast, treatment with the LE extract did not affect the expression of procollagen type 1 in HDF-n cells exposed to UVA irradiation. Thirteen phenolic compounds, including derivatives of shikonin and caffeic acid, were identified by ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. These results suggest that LE-derived extracts may protect oxidative-stress-induced skin aging by inhibiting degradation of skin collagen, and that this protection may derive at least in part from the antioxidant phenolics present in these extracts. Further studies are warranted to determine the potential utility of LE-derived extracts in both therapeutic and cosmetic applications.

Topics: Antioxidants; Caffeic Acids; Cell Line; Cell Survival; Collagen; Fibroblasts; Humans; Hydrogen Peroxide; Keratinocytes; Lithospermum; Matrix Metalloproteinase 1; Naphthoquinones; Oxidative Stress; Phenols; Plant Extracts; Skin; Skin Aging; Ultraviolet Rays

To study the effect of beta-hydroxyisovaleryl shikonin (beta-HIVS) combined cisplatin on activities of ovarian cancer cell line SKOV3 in vivo and its possible mechanisms.. Cells were divided into the blank control group and six beta-HIVS groups (2 - 30 micromol/L). Effect of beta-HIVS at different concentrations on the activities of ovarian cancer cell line SKOV3 was detected using MTT assay. SKOV3 cells were treated with cisplatin (10, 20, and 40 micromol/L) and beta-HIVS (0.25, 1, and 2.5 micromol/L) combined cisplatin. Effect of beta-HIVS combined cisplatin on the activities of ovarian cancer cell line SKOV3 was determined by MTT assay. The expression of Bcl-2 and Bax after treated by different concentrations of beta-HIVS was detected by Western blot.. The activities of SKOV3 were inhibited by different concentrations of beta-HIVS dose-dependently. The 50% inhibition rate (IC50) was 7.37 micromol/L. There was statistical difference in IC50 between each concentration beta-HIVS group and the blank control group (P < 0.05). There was statistical difference in IC50 between the beta-HIVS (1 and 2.5 micromol/L) combined cisplatin groups and the cisplatin group (P < 0.05, P < 0.01). The synergistic effect on beta-HIVS showed dose-dependent manner. Results of Western blot showed beta-HIVS at different concentrations (5, 7.5, and 10 micromol/L) could obviously up-regulate the expression level of Bax protein and inhibit the expression level of Bcl-2 protein, showing statistical difference when compared with the control group (P < 0.01). CONCLUSIONS; HIVS could obviously inhibit in vitro growth of SKOV3 in a dose-dependent manner. With the range of concentration, beta-HIVS showed synergetic effect with cisplatin. Besides, along with increasing beta-HIVS concentrations, the synergetic effect was more significant. The synergetic effect might accelerate the apoptosis of SKOV3 through up-regulating Bax expression and inhibiting Bcl-2 expression.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cisplatin; Female; Humans; Naphthoquinones; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2

To minimize the cytotoxicity of shikonin and alkannin that arises through the generation of reactive oxygen species (ROS) and alkylation of the naphthazarin ring, two series of novel core-scaffold-modified shikonin and alkannin derivatives were designed. These derivatives, which differ in their configurational and positional isomerism (R-, S-, and 2- and 6-isomers) were synthesized in high enantiomeric excess (>99 % ee). The selectivity of the dimethylated derivatives was significantly higher than the parent shikonin in vitro, but some side effects were still observed in vivo. Surprisingly, the dimethylated diacetyl derivatives with poor anticancer activity in vitro showed tumor-inhibiting effects similar to paclitaxel without any toxicity in vivo. The anticancer activity of these derivatives is in agreement with their low ROS generation and alkylating capacity, emphasizing their potential as prodrugs. This strategy provides means to address the nonspecific cytotoxicity of naphthazarin analogues toward normal cells.

Topics: Alkylation; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Drug Design; Female; Male; Mice; Microsomes, Liver; Naphthoquinones; Neoplasms; Prodrugs; Rats; Reactive Oxygen Species; Stereoisomerism; Structure-Activity Relationship; Transplantation, Heterologous; Transplantation, Homologous

Over the last few decades, food allergy (FA) has become a common disease in infants in advanced countries. However, anti-allergic medicines available in the market have no effect on FA, and consequently effective drug therapies for FA are not yet available. We have already demonstrated that mucosal mast cells play an essential role in the development of FA in a murine model. Thus, we screened many constituents from medicinal herbs for the ability to inhibit rat basophilic leukemia-2H3 mast-like cell degranulation, and found that shikonin, a naphthoquinone dye from Lithospermum erythrorhizon, exhibited the most potent inhibitory effect among them. Furthermore, shikonin extremely inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of tumor necrosis factor (TNF)-α mRNA expression in mucosal-type bone marrow-derived mast cells (mBMMCs). Global gene expression analysis confirmed by real-time PCR revealed that shikonin drastically inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of mRNA expression of the nuclear orphan receptor 4a family (Nr4a1, Nr4a2 and Nr4a3) in mBMMCs, and knockdown of Nr4a1 or Nr4a2 suppressed the IgE/antigen-induced upregulation of TNF-α mRNA expression. Computational docking simulation of a small molecule for a target protein is a useful technique to elucidate the molecular mechanisms underlying the effects of drugs. Therefore, the simulation revealed that the predicted binding sites of shikonin to immunophilins (cyclophilin A and FK506 binding protein (FKBP) 12) were almost the same as the binding sites of immunosuppressants (cyclosporin A and FK506) to immunophilins. Indeed, shikonin inhibited the calcineurin activity to a similar extent as cyclosporin A that markedly suppressed the IgE/antigen-enhanced mRNA expression of TNF-α and the Nr4a family in mBMMCs. These findings suggest that shikonin suppresses mucosal mast cell activation by reducing Nr4a family gene expression through the inhibition of calcineurin activity. Therefore, shikonin has therapeutic potential for the treatment of allergic diseases as a new calcineurin inhibitor.

Topics: Animals; Anti-Allergic Agents; Calcineurin Inhibitors; Cell Degranulation; Cyclosporine; Down-Regulation; Gene Knockdown Techniques; Lithospermum; Male; Mast Cells; Mice; Mice, Inbred BALB C; Molecular Docking Simulation; Naphthoquinones; Nuclear Receptor Subfamily 4, Group A, Member 1; Nuclear Receptor Subfamily 4, Group A, Member 2; Rats; RNA, Messenger; Tumor Necrosis Factor-alpha

Chemoprevention has been a pivotal and effective strategy during the skin cancer treatment. Using human skin normal and tumor samples, we demonstrated that both the expression and activity levels of pyruvate kinase M2 (PKM2) were higher in skin tumor tissues than normal tissues, suggesting that PKM2, one of important metabolic enzyme, might serve as a target for skin cancer prevention and/or therapy. Shikonin, a small-molecule active chemical, has been studied as an anti-cancer drug candidate in human cancer models. However, the mechanism of action and the chemopreventive potential of shikonin are unclear. Herein, we used the skin epidermal JB6 P+ cells and demonstrated that shikonin suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induced neoplastic cell transformation and PKM2 activation in the early stage of carcinogenesis. Mitochondrial functions were inhibited by TPA treatment, as indicated by reduced mitochondrial membrane potential and mitochondrial respiration, which were restored by shikonin. We also examined the levels of lactate as a glycolysis marker, and shikonin suppressed its increase caused by tumor promoter treatment. Modulation of cell metabolism by shikonin was associated with G2-M phase accumulation, and Fra-1 (a major subunit of activator protein 1 in skin tumorigenesis) downregulation. In addition, we demonstrated that AMP-activated protein kinase (AMPK), an energy sensor, which is inactivated by TPA, shikonin could reverse AMPK activity. These results suggest that shikonin bears chemopreventive potential for human skin cancers in which PKM2 is upregulated, which might be mediated by inhibiting oncogenic activation, PKM2 activation, and mitochondrial dysfunction.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Western; Carrier Proteins; Cell Adhesion; Cell Proliferation; Cell Respiration; Cell Transformation, Neoplastic; Cells, Cultured; Epidermis; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mitochondria; Naphthoquinones; Oxygen Consumption; Tetradecanoylphorbol Acetate; Thyroid Hormone-Binding Proteins; Thyroid Hormones

To search for compounds that disrupt binding of the EWS-FLI1 fusion protein to its cognate targets, we developed a homogeneous high-throughput proximity assay and screened 5200 small molecule compounds. Many well-known DNA-binding chemotherapeutic agents, such as actinomycin D, cisplatin, doxorubicin, daunorubicin, and epirubicin scored in the assay and not surprising also disrupted the binding of other transcription factors. Unexpectedly, we found that Shikonin, a natural product from the root of Lithospermum erythrorhizon, similarly disrupted protein-DNA interactions. Mechanistic studies demonstrated that Shikonin displaces SYBR green from binding to the minor groove of DNA and is able to inhibit topoisomerase mediated DNA relaxation. In cells, Shikonin blocked the binding of EWS-FLI1 to the NR0B1 promoter, and attenuated gene expression. Shikonin rapidly induced G2/M arrest and apoptosis in Ewing sarcoma cells. These results demonstrate that contrary to other purported mechanisms of action, Shikonin is a DNA-binding cytotoxic agent.

Topics: Antineoplastic Agents; Apoptosis; Biological Products; Cell Line, Tumor; DNA; DNA Cleavage; Dose-Response Relationship, Drug; Humans; Lithospermum; Naphthoquinones; Plant Roots; Structure-Activity Relationship

Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21(Cip1). In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21(Cip1) was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21(Cip1) have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21(Cip1) facilitated shikonin-induced apoptosis, implying that p21(Cip1) delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21(Cip1) to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21(Cip1) actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21(Cip1), leading to p38 MAPK activation, and finally, promoting apoptosis.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinase 5; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction

Integrin β1 facilitates cancer cell adhesion, migration and metastasis by activating intracellular signaling pathways including the ERK and PI3K signaling pathways. In previous studies, shikonin, an active naphthoquinone isolated from the Chinese medicine Zi Cao (gromwell), showed effective anticancer activity both in vivo and in vitro. However, the mechanisms underlying shikonin's anticancer activity are not fully elucidated. Increasing evidence indicates that shikonin inhibits tumor metastasis, but little is known about the effect of shikonin on lung cancer cells. To better understand the anti-metastatic role of shikonin in lung cancer, in this study we sought to investigate the effect of shikonin on lung cancer cell proliferation, adhesion to extracellular matrices (ECM), migration and invasion in non-small cell lung cancer A549 cells. We also sought to investigate the molecular mechanisms underlying shikonin's anticancer effects. Here we showed that when non-small cell lung cancer A549 cells were treated with shikonin for 24h, 8.0μM shikonin significantly inhibited cell proliferation, while cells treated with less than 2.0μM shikonin for 24h significantly suppressed cell adhesion to the ECM, invasion and migration in a dose-dependent manner. Moreover, real-time PCR and Western blot analysis showed that shikonin led to a reduction in the expression of integrin β1 at the mRNA and protein levels. Further elucidation of the mechanisms involved revealed that shikonin repressed the phosphorylation of extracellular signal-regulated kinase (ERK1/2). Taken together, our findings provide new evidence that shikonin suppresses lung cancer invasion and metastasis by inhibiting integrin β1 expression and the ERK1/2 signaling pathway.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Lung Neoplasms; MAP Kinase Signaling System; Naphthoquinones

We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

Topics: Animals; Apoptosis; Base Sequence; DNA Primers; Dopamine; Glutathione; Glutathione Peroxidase; HEK293 Cells; Humans; Naphthoquinones; Oxidopamine; PC12 Cells; Rats; Reverse Transcriptase Polymerase Chain Reaction

Shikonin, an analog of naphthoquinone pigments isolated from the root of Lithospermum erythrorhyzon, was recently reported to exert beneficial anti-inflammatory effects both in vivo and in vitro. The present study aimed to investigate the potential therapeutic effect of shikonin in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Dexamethasone was used as a positive control to evaluate the anti-inflammatory effect of shikonin in the study. Pretreatment with shikonin (intraperitoneal injection) significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, shikonin significantly reduced the concentrations of TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid induced by LPS. Compared with the LPS group, lung histopathologic changes were less pronounced in the shikonin-pretreated mice. Additionally, Western blotting results showed that shikonin efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that shikonin exerts anti-inflammatory properties in LPS-mediated ALI, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. Shikonin may be a potential agent for the prophylaxis of ALI.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Lipopolysaccharides; Lithospermum; Lung; Male; Mice; Mice, Inbred BALB C; Naphthoquinones; NF-kappa B; Organ Size; Plant Roots; Signal Transduction

In order to investigate the effects of fungal elicitor and macroporous adsorption resin on shikonin accumulation in hairy roots of arnebia euchroma (Royle) Johnst, we used spectrophotometry to determine the total naphthoquinone content of the hairy roots, by adding different volume ratio of Aspergillus niger elicitor, Aspergillus oryzae elicitor, and the macroporous resin into the M-9 liquid medium at different culture time. The results show that the total naphthoquinone content was 2.28 times higher than the control when we added mixed elicitors of Aspergillus niger and Aspergillus oryzae at the ratio of 2.5:50 in the 10th day of hairy roots cultivating. The total naphthoquinone content was 3.71 times higher than that of the control, when we added macroporous adsorption resin NKA-9. Aspergillus niger elicitor exhibited synergistic effect with Aspergillus oryzae elicitor to enhance the naphthoquinone. Also, the total naphthoquinone level was 4.17 times higher than that of the control by adding mixed fungal elicitor and macroporous adsorption resin NKA-9 in the bioreactor. Aspergillus oryzae and mixed elicitor could promote the hairy roots proliferation, and macroporous adsorption resin NKA-9 and mixed elicitor increased the total naphthoquinone content. In summary, the measure developed for Arnebia euchroma (Royle) Johnst hairy roots cultivating in bioreactors may potential for large-scale production of naphthoquinone.

Topics: Aspergillus niger; Boraginaceae; Fungal Proteins; Naphthoquinones; Plant Roots; Porosity; Resins, Synthetic

The intestinal barrier is a complex system with a dynamic structure that is designed for the maintenance of homeostasis in healthy individuals. Ulcerative colitis, one of the main manifestations of inflammatory bowel disease, is characterized by an inadequate and delayed wound healing. Shikonin, the active principle in the root of Lithospermum erythrorhizon, has demonstrated its ability to attenuate dextran sulfate sodium-induced ulcerative colitis in mice. Moreover, the root of L. erythrorhizon has been used in traditional Chinese medicine for treatment of burns, anal ulcers, hemorrhoids and skin wounds. However, the effect of shikonin on intestinal wound healing is unknown. Using an in vitro model for wound healing, we observed that shikonin enhances cell migration of intestinal epithelial cells through a mechanism that involves TGF-β1 induction. The combination of shikonin's anti-inflammatory activity together with its wound-healing properties makes it a great potential therapeutic agent for the treatment of injury associated with intestinal inflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Movement; Cell Survival; Intestines; Naphthoquinones; Rats; STAT3 Transcription Factor; Transcription Factor RelA; Transforming Growth Factor beta; Wound Healing

Shikonin (SHK), a natural naphthoquinone derived from the Chinese medical herb Lithospermum erythrorhizon, induces both apoptosis and necroptosis in several cancer cell lines. However, the detailed molecular mechanisms involved in the initiation of cell death are still unclear. In the present study, caspase-dependent apoptosis was induced by SHK treatment at 1μM after 6h in U937 cells, with increase in DNA fragmentation, generation of intracellular reactive oxygen species (ROS), fraction of cells with low mitochondrial membrane potential (MMP), and in the expression of BH3 only proteins Noxa and tBid. Interestingly, caspase-independent cell death was also detected with SHK treatment at 10μM, observed as increase in SYTOX® Green staining and release of lactate dehydrogenase (LDH). Necrostatin-1 (Nec-1) completely inhibited the SHK-induced leakage of LDH and SYTOX® Green staining. Cell permeable exogenous glutathione (GSH) completely inhibited 1μM SHK-induced apoptosis and converted 10μM SHK-induced necroptosis to apoptosis. Gene expression profiling revealed that 353 genes were found to be significantly regulated by 1μM and 85 genes by 10μM of SHK treatment, respectively. Among these genes, the transcription factor 3 (ATF3) and DNA-damage-inducible transcript 3 (DDIT3) were highly expressed at 1μM of SHK treatment, while tumor necrosis factor (TNF) expression mainly increased at 10μM treatment. These findings provide novel information for the molecular mechanism of SHK-induced apoptosis and necroptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Death; Drugs, Chinese Herbal; Gene Expression; Gene Expression Profiling; Glutathione; Humans; Naphthoquinones; Necrosis; Oxidative Stress; U937 Cells

Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.. Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.. Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.. We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Shape; Cell Survival; Glioma; Humans; Medicine, Chinese Traditional; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Rats; Reactive Oxygen Species; RNA-Binding Proteins

Shikonin is a traditional Oriental medical herb extracted from Lithospermum erythrorhizon. Many studies have shown that shikonin possesses anticancer ability against many different cancers, including hepatocellular carcinoma (HCC). Recently, tumor metastasis has been become an important clinical obstacle. However, the effect of shikonin on metastasis by HCC is unknown. The 50% inhibitory concentration (IC50) of shikonin on HCC cells was determined by an MTT assay and the xCELLigence biosensor system. The migratory ability of HCC cells was detected by a transwell migration assay and the xCELLigence biosensor system. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) expression levels were determined by Western blotting, and the activities of MMP-2 and -9 were determined by gelatin zymography. We found that IC50 values of HepJ5 and Mahlavu cells to shikonin treatment were around 2 μM. Exposure to a low dose of shikonin (0-0.4 μM) did not influence the survival of HCC cells. Interestingly, exposure to a low dose of shikonin inhibited the migratory ability on HepJ5 and Mahlavu cells. To further dissect the mechanism, we found that treatment with a low dose of shikonin reduced the activities and expression levels of MMP-2 and -9, which were correlated with the decreased cell migratory ability of HCC cells. In addition, we found a decrease of vimnetin expression, but no influence on the expression levels of N-cadherin, TWIST, or GRP78. In mechanism dissecting, we found that shikonin treatment may suppress the phosphorylation of AKT and then reduce the NF-κB (NF = nuclear factor) levels, but has no influence on the levels of c-Fos and c-Jun. Furthermore, we also found that shikonin may also reduce the phosphorylation of IκB. We concluded that a low dose of shikonin can suppress the migratory ability of HCC cells through downregulation of expression levels of vimentin and MMP-2 and -9. Our findings suggest that shikonin may be a new compound to prevent the migration of HCC cells.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Drugs, Chinese Herbal; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Humans; Lithospermum; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthoquinones; Neoplasm Metastasis

A series of shikonin derivatives, selectively acylated by various fluorinated carboxylic acids at the side chain of shikonin, were synthesized and their anticancer activity evaluated, in which eight compounds are reported for the first time. Among all the compounds tested, compound showed the most potent anticancer activity against B16-F10 (malignant melanoma cells), MG63 (human osteosarcoma cells), and A549 (lung cancer cells) with IC50 0.39 ± 0.01, 0.72 ± 0.04 and 0.58 ± 0.02 µmol/L. Docking simulation of compound was carried out to position into a tubulin active site to determine the probable binding conformation. All the results suggested that compound may be a potential anticancer agent.

Topics: Acylation; Antineoplastic Agents; Carboxylic Acids; Cell Cycle; Cell Line, Tumor; Chemistry Techniques, Synthetic; Drug Design; Humans; Naphthoquinones; Protein Multimerization; Protein Structure, Quaternary; Substrate Specificity; Tubulin

The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells.. To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0-2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway.. Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin.. These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway during the early stages of adipogenesis.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Animals; Down-Regulation; Lithospermum; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; Phosphorylation; Plant Extracts

Via studying the phenotype, growth curve and secondary metabolites of two kinds of suspension culture cell of Arnebia euchroma, the kinetics parameters of growth and accumulation of shikonin compounds in cell suspension culture of A. euchroma was obtained through simulating and modeling. This Study found that the red high-yielding one was a fine cell line for producing shikonin compounds, and the white low-yielding one may be a mutant. The first-order and second-order derivative of the fitting function were obtained by fitting the Logistic model of growth curve to get the growth rate and growth acceleration curve of the suspended cells. It is found that the best period to subculture was the 15th day cultured in fresh medium, and the best period of the induction process was the 13th-14th day. When compared the growth rate of the red line and the shikonin compounds accumulation curve, it is found that the rapid growth of the biomass of cells was not conducive to the synthesis and accumulation of shikonin compounds.

Topics: Boraginaceae; Cell Culture Techniques; Cell Proliferation; Naphthoquinones; Plant Cells

Molecularly Imprinted Polymers (MIPs) targeting shikonin, a potent antioxidant and wound healing agent, have been prepared using methacrylic acid (MAA) and 2-diethylaminoethyl methacrylate (DEAEMA) as functional monomers. An investigation of solution association between shikonin and both acidic and basic functional monomers by UV-vis titrations, suggested stronger affinity towards the basic functionality. Strong inhibition of the co-polymerisation reaction of such basic monomers was observed, but was overcome by reduction of the amount of template used during polymer synthesis. Polymer morphology was severely impacted by the template's radical scavenging behaviour as demonstrated by solid state NMR spectroscopy measurements. HPLC evaluation of the final materials in polar conditions revealed limited imprinting effects and selectivity, with the MAA polymers exhibiting marginally better performance. During application of the polymers as MI-SPE sorbents in non-polar solvents it was found that the DEAEMA based polymer was more selective towards shikonin compared to the MAA counterpart, while shikonin recoveries of up to 72% were achieved from hexane solutions of a commercial sample of shikonin, hexane extract of Alkanna tinctoria roots and a commercial pharmaceutical ointment.

Topics: Boraginaceae; Drugs, Chinese Herbal; Methacrylates; Molecular Imprinting; Naphthoquinones; Plant Extracts; Plant Roots; Solid Phase Extraction

Shikonin, which is an active naphthoquinone isolated from traditional Chinese herbal medicine Zi Cao, has been recently developed to use as an antitumor agent in colorectal cancer, melanoma, leukemia, breast cancer, and hepatocellular cancer. However, its antitumor effect in thyroid cancer remains largely unknown.. The aim of the study was to test the therapeutic potential of shikonin for thyroid cancer and explore the mechanisms underlying antitumor effects of shikonin.. We examined the effects of shikonin on proliferation, cell cycle, apoptosis, migration, invasion, and xenograft tumor growth in thyroid cancer cells and the effect of shikonin on proliferation of primary thyroid cancer cells.. Shikonin inhibited thyroid cancer cell proliferation in a dose- and time-dependent manner and induced cell cycle arrest. Moreover, shikonin induced cell apoptosis through reactive oxygen species-mediated DNA damage and activation of the p53 signaling pathway. Our data also showed that shikonin dramatically inhibited thyroid cancer cell migration and invasion by suppressing epithelial-mesenchymal transition and downregulating expression of Slug and MMP-2, -9, and -14. Further elucidation of the mechanisms involved revealed that shikonin markedly repressed the phosphorylation of Erk and Akt and activated the p16/Retinoblastoma protein (Rb) pathway in thyroid cancer cells. Growth of xenograft tumors derived from the thyroid cancer cell line FTC133 in nude mice was significantly inhibited by shikonin. Importantly, we did not find the effect of shikonin on liver function in mice.. We for the first time demonstrated that shikonin is a potentially effective antitumor agent for thyroid cancers.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Movement; Cyclin-Dependent Kinase Inhibitor p16; Epithelial-Mesenchymal Transition; Humans; Inhibitory Concentration 50; Liver; Mice; Mice, Nude; Mutant Proteins; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Proteins; Retinoblastoma Protein; Signal Transduction; Thyroid Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

The extraction of functional components from radix of Arnebia euchroma was optimized using orthogonal design based on the extraction yields of shikonin, and hydroxyl-naphthoquinone pigments. The data processing was carried out with the multiple guidelines grading method for optimizing the extraction condition. Compared with the traditional method (refluxing and ultrasonic extraction), the flash extraction method was more efficient The optimal conditions were as follows: 95% ethanol extract 3 times with 90 s for each. Under these conditions, the extraction yields of shikonin, and hydroxyl-naphthoquinone pigments were 93.16%, 93.89%, respectively, and the dry extract rate was 5.16%. In conclusion, the result showed that the flash extraction technology was appropriate, stable and feasible.

Topics: Boraginaceae; Drugs, Chinese Herbal; Naphthoquinones; Pigments, Biological; Plant Extracts

Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 μmol/L of shikonin and 3 μmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.

Topics: Apoptosis; Brain Neoplasms; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Glioma; Humans; Naphthoquinones; Neoplastic Stem Cells; Proto-Oncogene Proteins c-bcl-2; Topoisomerase I Inhibitors; Topotecan

Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers.. MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo.. The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001).. Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Naphthoquinones; Necrosis; Neoplasm Transplantation; Nuclear Pore Complex Proteins; Osteosarcoma; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Up-Regulation

Inducing apoptosis is an important and promising therapeutic approach to overcome cancer. Here, we described a series of novel synthesized compounds, cinnamic acyl shikonin derivatives (1b-19b), which were synthesized starting from shikonin and cinnamic acids, which exhibit anticancer activity via inducing apoptosis in vitro. Our flow cytometry results showed that compound 8b((E)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent -3-enyl-3-(3-(trifluoromethyl) phenyl)acrylate) (IC(50) = 0.69, 0.65, 1.62 μM for human SW872-s, A875 and A549 cell lines, respectively) exhibited conspicuous anticancer activities and has low cell toxicity in vitro. Therefore, we considered that compound 8b is potentially to be a candidate of anticancer agent. The proliferation inhibitory effect of compound 8b was associated with its apoptosis-inducing effect by activating caspase-3, caspase-7, caspase-9, and PARP. When the level of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved PARP are rise, apoptosis of cancer cells will be induced.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cinnamates; Humans; Naphthoquinones; Poly(ADP-ribose) Polymerases

Shikonin, a natural naphthoquinone pigment extracted from the root of Lithospermum erythrorhizon, has shown a variety of pharmacologic properties including anti-inflammatory effect. In the present study, we analyzed the role of shikonin in acute lung injury induced by lipopolysaccharide (LPS) in mice.. Sixty male BALB/C mice were randomly allocated into six groups (n = 10, each): control group, shikonin group (50 mg/kg), LPS group, and three different doses (12.5, 25, and 50 mg/kg) for shikonin-treated groups. Shikonin or vehicle was given with an intragastric administration 1 h before an intratracheal instillation of LPS (5 mg/kg). The severity of pulmonary injury was evaluated 6 h after LPS challenge.. Shikonin pretreatment significantly attenuated LPS-induced pulmonary histopathologic changes, alveolar hemorrhage, and neutrophil infiltration. The lung wet-to-dry weight ratios, as the index of pulmonary edema, were markedly decreased by shikonin pretreatment. Moreover, shikonin decreased the productions of the proinflammatory cytokines including tumor necrosis factor alpha and interleukin 1β and the concentration of total proteins in the bronchoalveolar lavage fluid. Shikonin pretreatment also reduced the concentrations of myeloperoxidase and nitric oxide in lung tissues. In addition, shikonin pretreatment significantly suppressed LPS-induced activation of cyclooxygenase 2 and inducible nitric oxide synthase and the nuclear factor κB DNA-binding activity in lung tissues.. This study indicates that shikonin may have a protective effect against LPS-induced acute lung injury, and the potential mechanism of this action may attribute partly to the inhibition of inducible nitric oxide synthase and cyclooxygenase 2 expression by downregulating nuclear factor κB activation.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Cyclooxygenase 2; DNA; Drugs, Chinese Herbal; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred BALB C; Naphthoquinones; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Peroxidase; Pulmonary Edema

Shikonin has anticancer activity, but it has not yet been applied into clinical use. In the present study, shikonin was prepared using liposomes. We aimed to examine several aspects of sh-L (shikonin-containing liposomes): preparation, angiogenic suppression and cellular uptake through self-fluorescence. Sh-L were prepared using soybean phospholipid and cholesterol to form the membrane and shikonin was encapsulated into the phospholipid membrane. Three liposomes were prepared with shikonin. They had red fluorescence and were analysed using a flow cytometer. Angiogenic suppression of sh-L was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], Transwell tests, chick CAM (chorioallantoic membrane) and Matrigel™ plug assay. MTT assay showed the median IC₅₀ (inhibitory concentrations) as follows: shikonin, sh-L₁ and sh-L₂ were 4.99±0.23, 5.81±0.57 and 7.17±0.69 μM, respectively. The inhibition rates of migration were 53.58±7.05, 46.56±4.36 and 41.19±3.59% for 3.15 μM shikonin, sh-L₁ and sh-L₂, respectively. The results of CAM and Matrigel plug assay demonstrated that shikonin and sh-L can decrease neovascularization. Effect of shikonin was more obvious than sh-L at the same concentration. The results showed that sh-L decreased the toxicity, the rate of inhibition of migration and angiogenic suppression. The cellular uptake of the sh-L could be pictured because of the self-fluorescence. The self-fluorescence will be useful for conducting further research. Sh-L might be an excellent preparation for future clinical application to cancer patients.

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Chickens; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Humans; Liposomes; Naphthoquinones; Neovascularization, Pathologic

Shikonin, a highly liposoluble naphthoquinone pigment isolated from the traditional medical herbs Lithospermum erythrorhizon (LE), was considered to exhibit an anti-inflammatory property. While the potential of shikonin to ameliorate acute pancreatitis (AP) is unknown. Our aim was to investigate the effects of shikonin in a murine model of cerulein-induced pancreatitis.. AP was induced in mice by six intraperitoneal injection of cerulein (50 μg/kg) at hourly intervals. Vehicle or shikonin (50 mg/kg) was pretreated 2 h before the first cerulein injection. After 6 h, 9 h and 12 h of the first cerulein injection, the severity of acute pancreatitis was assessed by biochemistry, myeloperoxidase activity, histological grading, proinflammatory cytokines levels and nuclear factor kappa B (NF-κB) activity.. Shikonin administration significantly reduced serum amylase and lipase activities, pancreatic histological scores, TNF-α, IL-1β, IL-6 levels, MPO activity and NF-κB activity.. Taken together, these results suggest that shikonin might protect against experimental pancreatitis by reducing release of inflammatory cytokines via inhibition of NF-κB activity. The therapeutic role of shikonin in AP needs further investigation.

Topics: Animals; Anti-Inflammatory Agents; Ceruletide; Cytokines; Male; Mice; Mice, Inbred BALB C; Naphthoquinones; NF-kappa B; Pancreatitis; Peroxidase; Phytotherapy; RNA, Messenger

An alternative cell demise programmed necrosis has also been proposed when apoptotic machinery is impaired or blocked during tumor necrosis factor alpha (TNFα) stimulation. Shikonin (SKN), an herbal extract from the Chinese plant, has been reported to induce either apoptosis or necrosis depending on cell types or its concentrations. In this presentation, SKN caused cell death of NIH3T3 in a dose-dependent manner. Intriguingly, SKN-mediated cell death was in part protected by necrostatin-1 (Nec-1), a specific inhibitor of programmed necrosis, but not zVAD a pan-caspase inhibitor. SKN directly mediated cell death via receptor interacting protein1 and 3 (RIP1-RIP3) complex formation, which is required for TNFα-mediated programmed necrosis. Additionally, SKN-caused cell death was reversed by a reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) whereas TNFα-mediated necrosis was successfully protected by butylated hydroxyanisole (BHA), implying that ROS may be differentially derived from death inducing agents. Concurrently with the protective effect of the ROS scavenger or Nec-1 on TNFα or SKN, the RIP1-RIP3 complex was significantly affected in the presence of those agents. Here, it is highlighted that SKN as well as TNFα can directly mediate cell death via a pronecrotic complex, but ROS were generated via different routes depending on cell death-inducing agents.

Topics: Acetylcysteine; Animals; Apoptosis; Mice; Naphthoquinones; NIH 3T3 Cells; Protein Binding; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases

An efficient homogenate extraction technique was employed for extracting shikonin from Arnebia euchroma. The homogenate extraction procedure was optimized and compared with other conventional extraction techniques. The proposed method gave the best result with the highest extraction efficiency in the shortest extraction time. Based on single-factor experiments, a three-factor-three-level experimental design has been developed by Box-Behnken design. The optimal conditions were 78% ethanol as solvent, homogenate extraction time of 4.2 min, 10.3 liquid to solid ratio and two extraction cycles. Moreover, the proposed method was validated by stability, repeatability and recovery experiments. The developed homogenate extraction method provided a good alternative for the extraction of shikonin from A. euchroma. The results indicated that the proposed homogenate extraction was a convenient, rapid and efficient sample preparation technique and was environmental friendly. Furthermore, homogenate extraction has superiority in the extraction of thermally sensitive compounds from plant matrices.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Drug Stability; Drugs, Chinese Herbal; Ethanol; Green Chemistry Technology; Models, Chemical; Models, Statistical; Naphthoquinones; Reproducibility of Results; Solid Phase Extraction; Solvents

Cancer drug resistance is a major obstacle for the success of chemotherapy. Since most clinical anticancer drugs could induce drug resistance, it is desired to develop candidate drugs that are highly efficacious but incompetent to induce drug resistance. Numerous previous studies have proven that shikonin and its analogs not only are highly tumoricidal but also can bypass drug-transporter and apoptotic defect mediated drug resistance. The purpose of this study is to investigate if or not shikonin is a weak inducer of cancer drug resistance.. Different cell lines (K562, MCF-7, and a MDR cell line K562/Adr), after repeatedly treated with shikonin for 18 months, were assayed for drug resistance and gene expression profiling.. After 18-month treatment, cells only developed a mere 2-fold resistance to shikonin and a marginal resistance to cisplatin and paclitaxel, without cross resistance to shikonin analogs and other anticancer agents. Gene expression profiles demonstrated that cancer cells did strongly respond to shikonin treatment but failed to effectively mobilize drug resistant machineries. Shikonin-induced weak resistance was associated with the up-regulation of βII-tubulin, which physically interacted with shikonin.. Taken together, apart from potent anticancer activity, shikonin and its analogs are weak inducers of cancer drug resistance and can circumvent cancer drug resistance. These merits make shikonin and its analogs potential candidates for cancer therapy with advantages of avoiding induction of drug resistance and bypassing existing drug resistance.

Topics: Antineoplastic Agents; Cell Proliferation; Cisplatin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; MCF-7 Cells; Naphthoquinones; Neoplasms; Paclitaxel; Time Factors

Shikonin, a major component of Lithospermum erythrorhizon and Arnebia euchroma, exhibits antiinflammatory, immunomodulatory and antitumour activities. Although many recent studies have focused on the antitumour effects of shikonin, the exact mechanisms underlying its antitumour and immunomodulatory effects in tumour-bearing mice remain unclear. The aim of the present study was to investigate the antitumour and immunomodulatory effects of shikonin derivatives (ShD) in tumour-bearing mice. Swiss mice inoculated with hepatoma HepA(22) or sarcoma 180 (S(180)) cells were treated with ShD or 5-fluorouracil (5Fu). Survival time, immune organs, natural killer cell activity, lymphocytes, lymphocyte transformation and interleukin (IL)-2 production were analysed. ShD significantly prolonged the survival (median survival time prolonged by >7 days) of tumour-bearing mice in a dose-dependent manner, inhibited the growth of transplantable neoplasms (inhibitory rate, > 33%), and recovered (at [ShD] = 2.5 mg/kg/day) or increased (at [ShD] > 5 mg/kg/day) the number of CD3- and CD19-positive cells. ShD also played a role in protecting the immune organs from damage and reversed or enhanced immune responses, as noted by the nearly normal thymic structure; enlarged splenic corpuscles; and improved natural killer cell activity, lymphocyte transformation and IL-2 production in ShD-treated mice. ShD reduced the tumour load of tumour-bearing mice and protected the immune organs against tumour-induced damage and immune function impairment.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD19; Antineoplastic Agents, Phytogenic; Boraginaceae; Carcinoma, Hepatocellular; CD3 Complex; Dose-Response Relationship, Drug; Fluorouracil; Interleukin-2; Killer Cells, Natural; Lithospermum; Liver Neoplasms; Lymphocytes; Mice; Naphthoquinones; Phytotherapy; Plant Extracts; Sarcoma; Spleen; Thymus Gland

The radical scavenging activity of shikonin and acylshikonin derivatives was studied by using density functional theory. The hydrogen bond property of the studied structures was investigated using the atoms in molecules (AIM) theory. It turned out that the hydrogen bond is important for good radical scavenging activity. The hydrogen atom transfer for shikonin and acylshikonin derivatives is difficult to obtain because of the high bond dissociation energy (BDE). However, shikonin and acylshikonin derivatives appear to be good candidates for the one-electron-transfer. The introduction of acyl groups for shikonin decreases the ionization potential (IP) values compared with that of shikonin. The acylshikonin derivatives with 1H-pyrrole, furan, and thiophene groups are expected to be of the highest radical scavenging activity among the compounds investigated in this study. Taking this system as an example, we present an efficient method for the investigation of radical scavenging activity from theoretical point of view.

Topics: Free Radical Scavengers; Free Radicals; Hydrogen Bonding; Models, Molecular; Molecular Structure; Naphthoquinones; Structure-Activity Relationship

The interest of drug delivery has focused on the creation of new formulations with improved properties, taking much attention to the drug release from the carrier. Liposomes have already been commercialized, while dendrimers and hyperbranched polymers are emerging as potentially ideal drug delivery vehicles. Chimeric advanced drug delivery nano systems (chi-aDDnSs) are mixed nanosystems combining different biomaterials that can offer advantages as drug carriers. Alkannin and shikonin (A/S) are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant and recently established antitumor activity. In this work three generations of hyperbranched aliphatic polyesters were used for the first time to form complexes with shikonin, as well as liposomal chi-aDDnSs. Characterization of the shikonin-loaded chi-aDDnSs was performed by measuring their particle size distribution, ζ-potential, drug encapsulation efficiency and the in vitro release profile. The analysis revealed sufficient drug encapsulation and appropriately featured release profiles. Chi-aDDnSs were also examined for their physical stability at 4°C. The results are considered promising and could be used as a road map for designing in vivo experiments.

Topics: Chemistry, Pharmaceutical; Dendrimers; Drug Carriers; Drug Compounding; Drug Stability; Drugs, Chinese Herbal; Kinetics; Liposomes; Molecular Structure; Nanotechnology; Naphthoquinones; Particle Size; Solubility; Solvents; Technology, Pharmaceutical; Temperature

Three new hydroquinone terpenoids with benzogeijerene skeletons, euchroquinols A-C (1- 3), and a new monoterpenylbenzenoid, 9,17-epoxyarnebinol (4), along with five known compounds were isolated from the stem bark of ARNEBIA EUCHROMA. Shikonin (6) exhibited potent anti-HCV activity with a selective index of 43.56, and compounds 1, 6, and des-O-methyllasiodiplodin (7) showed anti-Staphylococcus aureus activity with MICs of 0.5, 0.125, and 0.125 mg/mL, respectively.

Topics: Anti-Bacterial Agents; Antiviral Agents; Boraginaceae; Hepacivirus; Microbial Sensitivity Tests; Naphthoquinones; Plant Bark; Plant Extracts; Plant Stems; Staphylococcus aureus

Environmental stress induces damage that activates an adaptive response in any organism. The cellular stress response is based on the induction of cytoprotective proteins, the so-called stress or heat shock proteins (HSPs). HSPs are known to function as molecular chaperones which are involved in the therapeutic approach of many diseases. Therefore in the current study we searched nontoxic chaperone inducers in chemical compounds isolated from medicinal plants. Screening of 80 compounds for their Hsp70-inducing activity in human lymphoma U937 cells was performed by western blotting. Five compounds showed significant Hsp70 up-regulation among them shikonin was most potent. Shikonin was able to induce Hsp70 at 0.1 µM after 3 h without activation of heat shock transcription factor 1 (HSF-1). It also induces significant reactive oxygen species generation. The expression level of genes responsive to shikonin was studied using global-scale microarrays and computational gene expression analysis tools. Significant increase in the nuclear factor erythroid 2-related factor 2 (Nrf2, NFEL2L2) -mediated oxidative stress response was observed that leads to the activation of HSP. The results of gene chip analysis were further confirmed by real-time qPCR assay. In short, the detailed mechanisms of Hsp70 induction by shikonin is not fully understood, Nrf2 and its target genes might be involved in the Hsp70 up-regulation in U937 cells.

Topics: Apoptosis; Drugs, Chinese Herbal; Gene Expression Regulation; Heat-Shock Proteins; Hot Temperature; Humans; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Plants, Medicinal; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; U937 Cells

The N-acetyl glucosaminosides of shikonin/alkannin were synthesized by chemical glycosylation, which provided an ideal approach to resolve the mixture of enantiomeric shikonin and alkannin co-existed in the Chinese herbs. The glycosylated shikonin and alkannin exhibited stronger binding activity to human telomeric G-quadruplex DNA than their parent structures. This research indicated that glycosylation of natural product with amino sugars is an effective strategy to improve their DNA-binding affinity.

Topics: Aminoglycosides; DNA; G-Quadruplexes; Glycosylation; Humans; Molecular Structure; Naphthoquinones; Spectrometry, Mass, Electrospray Ionization

Sialidases are enzymes that catalyze the hydrolysis of sialic acid residues from various glycoconjugates, which are widely found in a number of viral and microbial pathogens. In this study, we investigated the biological evaluation of isolated six shikonins (1-6) and three shikonofurans (7-9) from Lithospermum erythrorhizon. The nine isolated compounds 1-9 showed strong and selective inhibition of glycosyl hydrolase (GH) 33 and -34 sialidases activities. In GH33 bacterial-sialidase inhibition assay, the inhibitory activities against GH33 siadliase of all shikonofuran derivatives (7-9) were greater than shikonin derivatives (1-6). Shikonofuran E (8) exhibited the most potent inhibitory activity toward GH33 sialidases (IC(50)=0.24μM). Moreover, our detailed kinetic analysis of these species unveiled that they are all competitive and simple reversible slow-binding inhibitors. Otherwise, they showed different inhibitory capacities and kinetic modes to GH34 viral-sialidase activity. All the naphthoquinone derivatives (1-6) were of almost equal efficiency with IC(50) value of 40μM and shikonofurans (7-9) did not show the significant inhibitory effect to GH34 sialidase. Kinetic analyses indicated that naphthoquinones acted via a noncompetitive mechanism.

Topics: Glycoside Hydrolases; Hydrolases; Kinetics; Lithospermum; Naphthoquinones; Neuraminidase

Shikonin is the main naphthoquinone compound of the root of Lithospermum erythrorhizon. Our previous study demonstrated that shikonin possesses anticancer activity in human hepatoma cells. However, the anticancer mechanism of shikonin in human glioma cells is unclear at present. In the present study, we demonstrated that shikonin induces apoptosis in three human glioma cell lines: U87MG, Hs683, and M059K cells.. Cell cycle, generation of reactive oxygen species (ROS), depletion of glutathione (GSH), and disruption of mitochondrial transmembrane potential in shikonin-treated cells were determined by flow cytometry. Apoptosis-related proteins, catalase, and superoxide dismutase-1 (SOD-1) were determined by Western blot testing. N-acetylcysteine (NAC), pifithrin-α (PFT-α), or cyclosporin A were applied to evaluate the molecular mechanism of shikonin in apoptosis.. Shikonin induces the generation of ROS, depletion of GSH, disruption of mitochondrial transmembrane potential, upregulation of p53, and cleavage of PARP [poly(ADP-ribose) polymerase] in U87MG glioma cells. Moreover, shikonin causes catalase downregulation and SOD-1 upregulation as well as decreased Bcl-2 and increased Bax expression. Pretreatment with NAC, PFT-α, or cyclosporin A causes the recovery of shikonin-induced apoptosis. The ROS generation and GSH depletion induced by shikonin trigger mitochondrial transmembrane potential disruption. ROS production was partially dependent on the upregulation of p53 upon shikonin treatment.. These studies are the first to show that shikonin-induced apoptosis occurs through multiple pathways in human glioma cells. We conclude that shikonin may be used as a potential chemotherapeutic agent against human glioma.

Topics: Acetylcysteine; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Catalase; Cell Line, Tumor; Cyclosporine; G1 Phase Cell Cycle Checkpoints; Glioma; Glutathione; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Oxidative Stress; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Superoxide Dismutase; Superoxide Dismutase-1; Toluene; Tumor Suppressor Protein p53

Shikonin, a phytochemical purified from Lithospermum erythrorhizon, has been shown to confer diverse pharmacological activities, including accelerating granuloma formation, wound healing, anti-inflammation and others, and is explored for immune-modifier activities for vaccination in this study. Transdermal gene-based vaccine is an attractive approach for delivery of DNA transgenes encoding specific tumor antigens to host skin tissues. Skin dendritic cells (DCs), a potent antigen-presenting cell type, is known to play a critical role in transmitting and orchestrating tumor antigen-specific immunities against cancers. The present study hence employs these various components for experimentation.. The mRNA and protein expression of RANTES were detected by RT-PCR and ELISA, respectively. The regional expression of RANTES and tissue damage in test skin were evaluated via immunohistochemistry assay. Fluorescein isothiocyanate sensitization assay was performed to trace the trafficking of DCs from the skin vaccination site to draining lymph nodes. Adjuvantic effect of shikonin on gene gun-delivered human gp100 (hgp100) DNA cancer vaccine was studied in a human gp100-transfected B16 (B16/hgp100) tumor model.. Among various phytochemicals tested, shikonin induced the highest level of expression of RANTES in normal skin tissues. In comparison, mouse RANTES cDNA gene transfection induced a higher level of mRANTES expression for a longer period, but caused more extensive skin damage. Topical application of shikonin onto the immunization site before gene gun-mediated vaccination augmented the population of skin DCs migrating into the draining lymph nodes. A hgp100 cDNA gene vaccination regimen with shikonin pretreatment as an adjuvant in a B16/hgp100 tumor model increased cytotoxic T lymphocyte activities in splenocytes and lymph node cells on target tumor cells.. Together, our findings suggest that shikonin can effectively enhance anti-tumor potency of a gene-based cancer vaccine via the induction of RANTES expression at the skin immunization site.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cancer Vaccines; Cell Line, Tumor; Chemokine CCL5; Dendritic Cells; gp100 Melanoma Antigen; Humans; Male; Mice; Naphthoquinones; Skin Neoplasms; Vaccines, DNA

Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.

Topics: Animals; Apoptosis; B7-2 Antigen; Cancer Vaccines; Cells, Cultured; Dendritic Cells; Female; Genes, MHC Class II; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Naphthoquinones; Skin Neoplasms; Spleen; Th1 Cells; Th17 Cells

To explore the mechanism of shikonin for inducing the apoptosis of human promyelocytic leukemia cell HL-60.. The effects of shikonin on the HL-60 cell proliferation were detected using MTT. The apoptosis rate was analyzed by Annexin-V/PI double staining. The expression level of the bcl-2 gene was detected using semi-quantitative reverse transcriptase PCR (RT-PCR), thus analyzing the correlation between the bcl-2 expression level and the apoptosis of HL-60.. Shikonin could inhibit the proliferation of HL-60 cells with the concentration range of 1-8 microg/mL in a time- and concentration-dependent manner. Two microg/mL shikonin could induce the apoptosis of HL-60 cells in a time-dependent manner. The expression level of bcl-2 was obviously down-regulated at 2 microg/mL shikonin.. Shikonin could induce the apoptosis of HL-60 cells. Its mechanism was correlated with down-regulation of the expression level of bcl-2.

Topics: Apoptosis; Cell Proliferation; HL-60 Cells; Humans; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2

Topics: Anthraquinones; Drugs, Chinese Herbal; Ions; Naphthoquinones; Pentanoic Acids; Spectrometry, Mass, Electrospray Ionization

Necrostatin-1 (Nec-1) inhibits necroptosis by allosterically inhibiting the kinase activity of receptor-interacting protein 1 (RIP1), which plays a critical role in necroptosis. RIP1 is a crucial adaptor kinase involved in the activation of NF-κB, production of reactive oxygen species (ROS) and the phosphorylation of mitogen activated protein kinases (MAPKs). NF-κB, ROS and MAPKs all play important roles in apoptotic signaling. Nec-1 was regarded as having no effect on apoptosis. Here, we report that Nec-1 increased the rate of nuclear condensation and caspases activation induced by a low concentration of shikonin (SHK) in HL60, K562 and primary leukemia cells. siRNA-mediated knockdown of RIP1 significantly enhanced shikonin-induced apoptosis in K562 and HL60 cells. Shikonin treatment alone could slightly inhibit the phosphorylation of ERK1/2 in leukemia cells, and the inhibitory effect on ERK1/2 was significantly augmented by Nec-1. We also found that Nec-1 could inhibit NF-κB p65 translocation to the nucleus at a later stage of SHK treatment. In conclusion, we found that Nec-1 can promote shikonin-induced apoptosis in leukemia cells. The mechanism by which Nec-1 sensitizes shikonin-induced apoptosis appears to be the inhibition of RIP1 kinase-dependent phosphorylation of ERK1/2. To our knowledge, this is the first study to document Nec-1 sensitizes cancer cells to apoptosis.

Topics: Active Transport, Cell Nucleus; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Nucleus; HL-60 Cells; Humans; Imidazoles; Indoles; K562 Cells; Leukemia; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; Nuclear Pore Complex Proteins; RNA-Binding Proteins; Transcription Factor RelA

To compare the composition and content of Arnebiae Radix and the stem residues.. TLC and HPLC were used to identify Arnebia, ultraviolet-visible spectrophotometry was used to determine the content of hydroxy naphthoquinone total pigment in Arnebia, HPLC was used to determine the total content of /3-P'-dimethyl acrylamide Aka Ning and shikonin.. The number of spots of Arnebia Radix was consistent with that of the stem residues in 10 batches of medicinal materials, the former was larger and deeper in color. Their features of fingerprint are the same,at the same retention time,the peak area of radix was larger; The average content of hydroxy naphthoquinone total pigment was 3.631% in the radix, and 1.516% in the stem. The total content of beta-beta'-dimethyl acrylamide Aka Ning and shikonin in the radix and the stem were respectively 0.89% and 0.309%.. The ingredients in the radix and the stem residues are the same, but the contents of root of the total pigment content of hydroxyl-naphthoquinone, beta-beta'-dimethyl acrylamide Aka Ning and shikonin are twice higher than those of the stem residues.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Boraginaceae; China; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Naphthoquinones; Plant Roots; Plant Stems; Plants, Medicinal; Quality Control; Reproducibility of Results; Solvents; Spectrophotometry, Ultraviolet

Lithospermum erythrorhizon has long been used in traditional Asian medicine for the treatment of diseases including skin cancer. In this study, hexane extract from the roots of Lithospermum erythrorhizon (LEH) was chemically characterized and its anticancer activity was tested against the most aggressive form of skin cancer.. The in vitro anticancer studies viz. cell growth, cell cycle and apoptosis, and the expression of tumor regulating proteins were analyzed against B16F10 melanoma cells. In addition, C57BL/6 mice models were used to evaluate the in vivo anticancer potential of LEH. Mice were intraperitoneally injected with LEH at doses of 0.1 and 10mg/kg every 3 days. The tumor inhibition ratio was determined after 21 days of treatment and the histopathological analyses of the tumor tissues were compared. Further, LEH was purified and its active compounds were structurally elucidated and identified by NMR spectra and quantified by HPLC analyses.. LEH effectively inhibits the growth of melanoma cells with an IC(50) of 2.73μg/ml. Cell cycle analysis revealed that LEH increased the percentage of cells in sub-G1 phase by dose dependent manner. LEH exhibited down regulation of anti-apoptotic Bcl-2 family proteins and up regulation of apoptotic Bax protein expression. Importantly, LEH induced cleavage of poly (ADP-ribose) polymerase (PARP) and activated the caspase cascade (caspase 3) with this cleavage mediating the apoptosis of B16F10 cells. LEH treatment at a dose of 10mg/kg for 21 days in experimental mice implanted with tumors resulted in significant reduction of the tumor growth (43%) and weight (36%). Histopathology analysis of LEH treated tumor tissues showed evidence of increased necrotic cells in a concentration dependent manner. Meanwhile, five naphthoquinone compounds [Shikonin (1); Deoxyshikonin (2); β-Hydroxyisovalerylshikonin (3); Acetylshikonin (4) and Isobutyrylshikonin (5)] were purified from LEH and responsible for its anticancer activity.. LEH induced apoptosis in B16F10 cells by activation of caspase 3 and inducing sub-G1 cell cycle arrest. LEH exhibited both in vitro and in vivo anticancer activity. Shikonin derivatives in the LEH are responsible for the anticancer activity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Caspases; Female; G1 Phase Cell Cycle Checkpoints; Lithospermum; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Naphthoquinones; Phytotherapy; Plant Extracts; Plant Roots; Tumor Burden

Shikonin and its ester derivatives belong to a group of secondary metabolites with a wide array of beneficial effects on human health. However, shikonin is principally used in oil-based preparations due to the low solubility of the pigment in aqueous media, and the positive properties of shikonin are not exploited to their full potential. Such low aqueous solubility often results in poor bioavailability, makes shikonin undesirable for oral administration, and restricts its broadened use in the food and pharmaceutical industries. The purpose of this study was to enhance the aqueous solubility of shikonin by the addition of β-lactoglobulin and to characterize the macromolecule-ligand binding interaction by means of spectrophotometry, spectrofluorometry, high-performance liquid chromatography, and mass spectrometry. In the presence of β-lactoglobulin the solubility of shikonin is increased up to 181-fold. One shikonin molecule binds covalently to β-lactoglobulin via Cys(121), whereas the remaining pigment molecules most probably bind to the protein via noncovalent interactions.

Topics: Drug Compounding; Lactoglobulins; Naphthoquinones; Protein Binding; Solubility

Among various cancer cell lines, the leukemia cell line HL-60 was most sensitive to Shikonin, with evidence showing both the prooxidative activities and proapoptotic effects of micromolar concentrations of Shikonin. However, the mechanism involved in the cytotoxicity of Shikonin in the submicromolar range has not been fully characterized. Using biochemical and free radical biological experiments in vitro, we identified the prodifferentiated profiles of Shikonin and evaluated the redox homeostasis during HL-60 differentiation. The data showed a strong dose-response relationship between Shikonin exposure and the characteristics of HL-60 differentiation in terms of morphology changes, nitroblue tetrazolium (NBT) reductive activity, and the expression level of surface antigens CD11b/CD14. During drug exposure, intercellular redox homeostasis changes towards oxidation are necessary to support Shikonin-induced differentiation, which was proven by additional enzymatic and non-enzymatic redox modulators. A statistically significant and dose-dependent increase (P < 0.05) was recorded with regard to the unique expression levels of the Nrf2/ARE downstream target genes in HL-60 cells undergoing late differentiation, which were restored with further antioxidants employed with the Shikonin treatment. Our research demonstrated that Shikonin is a differentiation-inducing agent, and its mechanisms involve the Nrf2/ARE pathway to modulate the intercellular redox homeostasis, thus facilitating differentiation.

Topics: Antioxidants; CD11b Antigen; Cell Differentiation; Cell Proliferation; Cell Shape; Extracellular Space; HL-60 Cells; Homeostasis; Humans; Lipopolysaccharide Receptors; Models, Biological; Naphthoquinones; NF-E2-Related Factor 2; Nitroblue Tetrazolium; Oxidation-Reduction; Response Elements; RNA, Messenger; Signal Transduction

Our study showed that Shikonin (SK) could provide an action against almost all Candida albicans isolates tested. More importantly, to some Fluconazole (FCZ)-resistant Candida albicans, the action of SK (MIC(80) value 4 µg/mL) was shown to be >16 times higher than that of FCZ (MIC(80) >64 µg/mL). To clarify the mechanism underlying this action, we performed a comparative study in untreated control C. albicans and C. albicans treated with SK. In this study, we found that SK treatment increased generation of endogenous reactive oxygen species (ROS) and decreased mitochondrial membrane potential. Furthermore, anti-oxidants N-acetylcysteine (NAC) and glutathione (GSH) could reduce the antifungal activity of SK significantly in C. albicans. Our analyses also identified 9 differentially expressed genes, which were related to glycolysis-related genes (CDC19 and HXK2), fermentation-related genes (ALD5 and ADH1), antioxidant defense-related genes (SOD2 and SOD5), thioredoxin reductase-related gene (TRR1), mitochondrial respiratory electron transport chain-related gene (MRF1) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductase-related gene (EBP1). These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the action of SK against C. albicans.

Topics: Acetylcysteine; Antifungal Agents; Candida albicans; Gene Expression Regulation, Fungal; Genes, Fungal; Glutathione; Membrane Potential, Mitochondrial; Microbial Sensitivity Tests; Naphthoquinones; Reactive Oxygen Species

Cell suspension cultures of Arnebia euchroma were raised from in vitro leaf-derived friable callus on liquid MS [Murashige and Skoog] medium supplemented with BAP (6-benzylaminopurine) (10.0 μM) and IBA (indole-3-butyric acid) (5.0 μM). A two-stage culture system was employed using growth and production medium for cell biomass and shikonin derivatives, respectively. Factors such as light, temperature, sucrose and pH (hydrogen ion concentration) were studied to observe their effect on the shikonin derivative production. Light conditions completely inhibited shikonin derivative production. Out of different temperature regimes tested, the highest yield (586.17 μg/g FW) was found at 25°C. Maximum production (656.14 μg/g FW) was observed in 6% sucrose. An alkaline pH (7.25-9.50) favoured shikonin derivative production. The results showed that physical and chemical factors greatly influence the production of shikonin derivatives in cell suspension cultures of A. euchroma. Therefore, by employing optimum culture conditions, it is possible to enhance the production of secondary compounds from the cells. The factors optimized for in vitro production of shikonin derivatives during the present study can successfully be employed for their large-scale production in bioreactors.

Topics: Benzyl Compounds; Boraginaceae; Cell Culture Techniques; Cells, Cultured; Hydrogen-Ion Concentration; Indoles; Kinetin; Light; Naphthoquinones; Plant Leaves; Plants, Medicinal; Purines; Sucrose; Temperature

The aim of this study was to examine the anti-invasion effect of Shikonin on human high-metastatic adenoid cystic carcinoma (ACC-M) cells and to explain the possible molecular mechanism involved.. The ACC-M cells were treated with Shikonin (0, 2.5, 5, 10 μM) for 24 h. The protein levels and gelatinolytic activities of MMP-2 and MMP-9 were analyzed using Western blot and Gelatin zymography test, respectively. Matrigel invasion assays were used to investigate tumor invasive potential and electromobility shift assays were used to determine the activity of NF-κB.. The invasiveness of ACC-M cells was reduced in a dose dependent manner following 24-h treatment of up to 10 μM of the Shikonin at which concentration no cytotoxicity occurred. The protein levels and gelatinolytic activities of MMP-9 were significantly suppressed by increasing Shikonin concentrations. The down-regulation of MMP-9 appeared to be via the inactivation of NF-κB as the treatment with Shikonin suppressed the protein level of phosphate-IkBa, which was accompanied by a decrease in DNA-binding level of the factor.. Shikonin inhibits tumor invasion via downregulation of MMP-9 expression in ACC-M cells. Pharmacologic inhibition of the NF-κB-mediated MMP-9 expression by Shikonin might be a powerful treatment option for ACC patients in future.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Adenoid Cystic; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; Down-Regulation; Drugs, Chinese Herbal; Electrophoresis, Polyacrylamide Gel; Humans; I-kappa B Proteins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthoquinones; Neoplasm Invasiveness; NF-kappa B; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Necrosis Factor-alpha

Our previous study showed for the first time that shikonin, a natural compound isolated from Lithospermun erythrorhizon Sieb. Et Zucc, inhibits adipogenesis and fat accumulation. This study was conducted to investigate the molecular mechanism of the anti-adipogenic effects of shikonin.. Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate the crucial role of β-catenin in the anti-adipogenic effects of shikonin.. Shikonin prevented the down-regulation of β-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. β-catenin was a crucial mediator of the anti-adipogenic effects of shikonin, as determined by siRNA-mediated knockdown. Shikonin-induced reductions of the major transcription factors of adipogenesis including peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α, and lipid metabolizing enzymes including fatty acid binding protein 4 and lipoprotein lipase, as well as intracellular fat accumulation, were all significantly recovered by siRNA-mediated knockdown of β-catenin. Among the genes located in the WNT/β-catenin pathway, the levels of WNT10B and DVL2 were significantly up-regulated, whereas the level of AXIN was down-regulated by shikonin treatment.. This study clearly shows that shikonin inhibits adipogenesis by the modulation of WNT/β-catenin pathway in vitro, and also suggests that WNT/β-catenin pathway can be used as a therapeutic target for obesity and related diseases using a natural compound like shikonin, even though the in vivo effects of shikonin and its clinical significance remain to be elucidated.

Topics: 3T3-L1 Cells; Adipogenesis; Animals; beta Catenin; Blotting, Western; Gene Expression Regulation; Gene Knockout Techniques; Mice; Naphthoquinones; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Wnt Proteins

Tigloylshikonin, a new shikonin derivative esterified with tiglic acid ((E)-2-methylbut-2-enoic acid), was isolated as a minor pigment from a food colorant "Shikon color," a commercial root extract from Lithospermum erythrorhizon SIEBOLD et ZUCCARINI. The structure of tigloylshikonin was elucidated using (1)H, (13)C, the difference nuclear Overhauser effect (NOE), and 2D NMR techniques. Its stereochemistry was determined by chiral-phase HPLC analysis. Tigloylshikonin was also found in the roots of L. erythrorhizon, which indicated that this new shikonin derivative is a typical component of naphthoquinone pigments in the roots of L. erythrorhizon.

Topics: Chromatography, Liquid; Lithospermum; Magnetic Resonance Spectroscopy; Molecular Conformation; Naphthoquinones; Plant Roots; Spectrometry, Mass, Electrospray Ionization

Shikonin (β-alkannin), a naphthazarin derivative, has shown a variety of abilities such as anti-inflammatory, antitumoral, cytotoxic, and antimicrobial activities. In the presence of Cu(II), shikonin caused breakage of supercoiled plasmid pBR322 DNA. Other metal ions tested [Mg(II), Ca(II), and Ni(II)] were ineffective and only Fe(II) has the same ability in the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, catalase, thiourea, sodium azide, potassium iodide, and sodium benzoate. Cu(I) was shown to be an essential intermediate using the Cu(I)-specific sequestering reagent neocuproine. Shikonin induced HeLa cell apoptosis involved in the mechanism of increasing intracellular reactive oxygen species (ROS). It was suggested that shikonin generated ROS as a pro-oxidant in the presence of Cu(II), and ROS resulted in DNA damage and apoptotic cell death in cells.

Topics: Apoptosis; Copper; DNA Damage; Drugs, Chinese Herbal; HeLa Cells; Humans; Molecular Structure; Naphthoquinones; Oxidation-Reduction; Plasmids; Reactive Oxygen Species

This work systematically investigated the enantiomeric excess (e.e.) of main components isolated from the roots of three endemic Boraginaceae plants distributed extensively in China, named Arnebia euchroma (Royle) Johnst (A.e.), Lithospermum erythrorhizon Sieb. et Zucc. (L.e.) and Onosma confertum W. W. Smith (O.c.), and the optical purity of their hydrolysis products separately, by means of three different approaches. The influence of HCl on the e.e. values of the major constituents was also studied. Analysis of the absolute configurations and e.e. values of all the derivatives acquired was performed by CD and chiral-HPLC respectively. The results of the main constituents demonstrated that A.e. mainly yields S-form naphthoquinone derivatives, while the R-form is predominant in the derivatives of L.e. and O.c. The optical purity of alkannin and shikonin and their derivatives was not influenced by acid treatment in the course of separation and hydrolysis. Additionally, it was found that 100% e.e. of shikinon could be acquired from a specific shikinon ester derivative, β,β-dimethylacrylshikonin occurring in the roots of O.c., as did 100% e.e. of alkannin from β,β-dimethylacrylalkannin contained in the roots of A.e.

Topics: Boraginaceae; China; Chromatography, High Pressure Liquid; Circular Dichroism; Naphthoquinones; Plant Extracts; Plant Roots; Stereoisomerism

We previously showed that ethylene might be involved in the process of shikonin biosynthesis regulated by light signals. Here, we cloned a full-length cDNA of LeERF-1, a putative ethylene response factor gene, from Lithospermum erythrorhizon using the RACE (rapid amplification of cDNA ends) method. Phylogenetic analysis revealed that LeERF-1 was classified in the B3 subfamily, together with ERF1 and ORA59 of Arabidopsis. Heterologous expression of LeERF-1 in Arabidopsis showed that LeERF-1:eGFP fusion protein was precisely localised to the nucleus, implying that it might function as a transcription factor. Detailed expression analysis with real-time PCR showed that LeERF-1 was significantly down-regulated by white, blue and red light, although the inhibitory effect of red light was relatively weak compared to other light conditions. Tissue-specific expression analysis also indicated that LeERF-1 was dominantly expressed in the roots, which grow in soil in darkness. These patterns are all consistent with the effects of different light signals on regulating formation of shikonin and its derivatives, indicating that LeERF-1 might be a crucial positive regulator, like other B3 subfamily proteins (such as ORCA3 and ORA59), in regulating biosynthesis of secondary metabolites.

Topics: Arabidopsis; Base Sequence; Cloning, Molecular; DNA, Complementary; DNA, Plant; Down-Regulation; Gene Expression Regulation, Plant; Genes, Regulator; Light; Lithospermum; Molecular Sequence Data; Multigene Family; Naphthoquinones; Plant Proteins; Plants, Genetically Modified; Recombinant Fusion Proteins; Sequence Alignment; Transcription Factors

Alkannin, shikonin (A/S) and their derivatives are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant and antitumor activity. Clinical studies over the years revealed that A/S derivatives-based wound healing preparations (such as HELIXDERM(®)) are among a very small group of therapeutics that modulate both the inflammatory and proliferative phases of wound healing and present significant tissue regenerative activity. The purpose of the present work was to combine the biological properties of A/S and the advantages of electrospun meshes to prepare a potent topical/transdermal biomaterial for A/S. Four biocompatible polymers (cellulose acetate, poly(L-lactide), poly(lactide-co-glycolide) LA/GA:50/50 and 75/25) were used for the first time, to produce electrospun fiber mats containing either shikonin or A/S mixture in various amounts. Both drugs were effectively loaded into the above biomaterials. The incorporation of drugs did not considerably affect fibers morphology and their mean diameter size varied from 315 to 670 nm. High drug entrapment efficiencies (ranged from 74% to 95%) and appropriate release profiles were achieved, that render these fibers as potential A/S topical/transdermal wound healing dressings. Given the multifunctional activity of the natural products alkannins and shikonins, their consideration as bioactive constituents for tissue engineering scaffolds seems a promising strategy for repairing and regenerating tissues and mainly skin.

Topics: Administration, Cutaneous; Bandages; Cellulose; Drug Combinations; Drugs, Chinese Herbal; Electrochemical Techniques; Excipients; Humans; Lactic Acid; Naphthoquinones; Polyesters; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Wound Healing

Shikonin, extracted from medicinal Chinese herb (Lithospermum erythrorhizo), was reported to exert anti-inflammatory and anti-cancer effects both in vitro and in vivo. We have found that proteasome was a molecular target of shikonin in tumor cells, but whether shikonin targets macrophage proteasome needs to be investigated. In the current study, we report that shikonin inhibited inflammation in mouse models as efficiently as dexamethasone. Shikonin at 4 μM reduced the Lipopolysaccharides (LPS)-mediated TNFα release in rat primary macrophage cultures, and blocked the translocation of p65-NF-κB from the cytoplasm to the nucleus, associated with decreased proteasomal activity. Consistently, shikonin accumulated IκB-α, an inhibitor of NF-κB, and ubiquitinated proteins in rat primary macrophage cultures, demonstrating that the proteasome is a target of shikonin under inflammatory conditions. Shikonin also induced macrophage cell apoptosis and cell death. These results demonstrate for the first time that proteasome inhibition by shikonin contributes to its anti-inflammatory effect. The novel finding about macrophage proteasome as a target of shikonin suggests that this medicinal compound has great potential to be developed into an anti-inflammatory agent.

Topics: Active Transport, Cell Nucleus; Animals; Anti-Inflammatory Agents; Capillary Permeability; Cell Death; Cell Nucleus; Cytoplasm; Drugs, Chinese Herbal; Ear Auricle; Inflammation; Lithospermum; Macrophages; Mice; Naphthoquinones; NF-kappa B; Protease Inhibitors; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

Radix Lithosperm eyrthrorhizon is a common prescription compound in traditional Chinese medicine. Shikonin is a major component of Radix Lithospermi and has various biological activities. We have investigated the inhibitory effect of shikonin on the growth of adenovirus type 3 (AdV3) in vitro. The antiviral function of shikonin against AdV3 and its virus inhibition ratio were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (MTT). The expression of hexon protein in AdV3 was determined by immunofluorescence assay using laser scanning confocal microscopy (LSCM) and Western blot analysis. In addition, the rate of apoptosis in cells infected by AdV3 was determined by flow cytometry. Shikonin (0.0156-1 µM) inhibited growth of AdV3 in a concentration-dependent manner with a virus inhibition rate of 23.8-69.1%. Expression of hexon protein in AdV3 was higher in the virus control group than in the shikonin-treated groups as determined by immunofluorescence assay and Western blotting (p<0.05). The rate of shikonin-treated HeLa cell apoptosis had a statistically significant decrease with increasing concentration of drug (p<0.05). Our data demonstrate that shikonin possesses anti-AdV3 capabilities and that the potential antiviral mechanism might involve inhibiting the degree of apoptosis and hexon protein expression of AdV.

Topics: Adenoviridae; Adenoviridae Infections; Antiviral Agents; Apoptosis; Capsid Proteins; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; HeLa Cells; Humans; Lithospermum; Naphthoquinones; Phytotherapy; Plant Roots

Shikonin, an active component of Lithospermum erythrorhizon Sieb. et Zucc., shows multiple pharmacological properties. However, the estrogenic activity of shikonin is remaining unclear. We assessed the potential estrogenic activity of shikonin with dual-luciferase reporter assay and bioluminescent measurements, by using transient cotransfection with estrogen dependent plasmid pERE-TK-Luc and internal control plasmid pRL-TK in MCF-7 cells. Estrogenic activity of shikonin, even at high concentration did not alter significantly compared to negative control (p > 0.05) and were significantly lower than those with E2 (p < 0.01). Concluding, shikonin demonstrates no estrogenic activity in vitro.

Topics: Cell Line, Tumor; Genes, Reporter; Humans; Indicators and Reagents; Luciferases; Luminescence; Medicine, Chinese Traditional; Naphthoquinones; Phytoestrogens; Plasmids; Transfection

We recently reported that shikonin and its analogs were a class of necroptotic inducers that could bypass cancer drug resistance. However, the molecular targets of shikonin are not known. Here, we showed that shikonin and its analogs are inhibitors of tumor-specific pyruvate kinase-M2 (PKM2), among which shikonin and its enantiomeric isomer alkannin were the most potent and showed promising selectivity, that is, shikonin and alkannin at concentrations that resulted in over 50% inhibition of PKM2 activity did not inhibit PKM1 and pyruvate kinase-L (PKL). Shikonin and alkannin significantly inhibited the glycolytic rate, as manifested by cellular lactate production and glucose consumption in drug-sensitive and resistant cancer cell lines (MCF-7, MCF-7/Adr, MCF-7/Bcl-2, MCF-7/Bcl-x(L) and A549) that primarily express PKM2. HeLa cells transfected with PKM1 showed reduced sensitivity to shikonin- or alkannin-induced cell death. To the best of our knowledge, shikonin and alkannin are the most potent and specific inhibitors to PKM2 reported so far. As PKM2 universally expresses in cancer cells and dictates the last rate-limiting step of glycolysis vital for cancer cell proliferation and survival, enantiomeric shikonin and alkannin may have potential in future clinical application.

Topics: Antineoplastic Agents; Cell Line, Tumor; Enzyme Inhibitors; Glucose; Glycolysis; Humans; Lactic Acid; Naphthoquinones; Neoplasms; Pyruvate Kinase

We employ fully atomistic molecular dynamics simulations to study in detail the mechanisms involved in the non-covalent association of the bioactive agent Shikonin with the commercially available hyperbranched polyesters (Boltorn®), in ethanol solutions. We examine effects of the (pseudo)generation of the hyperbranched polyester and mimic two different concentrations, under conditions corresponding to excess drug availability. The two mechanisms participating in the polymer/drug complexation are hydrogen bonding and spatial constriction of the drug molecules within the hyperbranched structure. Based on static, as well as on dynamic information obtained by the analysis performed, it is demonstrated that apart from the size of the polyester, factors like the degree of structural flexibility, the intrapolymer hydrogen bonding and the polymer concentration may affect decisively the polyester/shikonin associative behavior, as well as the behavior of the drug-molecules in the solution. The results from the present study offer a detailed picture of the relative importance of those parameters affecting the complexation, and may serve as a basis for the understanding of the behavior of more complex multi-polyester systems.

Topics: Anti-Infective Agents; Antineoplastic Agents; Hydrogen Bonding; Molecular Conformation; Naphthoquinones; Polyesters; Solutions

Shikonin and β,β'-dimethylacrylshikonin in Arnebia euchroma (Royle) Johnst. were extracted by ionic liquid-based ultrasonic-assisted extraction (IL-based UAE) and determined by high-performance liquid chromatography (HPLC). The dried powder of A. euchroma (Royle) Johnst. was mixed with a room temperature ionic liquid [C(6)MIM][BF(4)] to form a suspension, and then the ultrasonic extraction was performed in a water bath at ambient temperature. The calibration curve showed good linear relationship (r>0.9998) in the concentration range of 1.75-140 μg/mL for shikonin and 2.15-1360 μg/mL for β,β'-dimethylacrylshikonin. The recoveries were between 69.79% and 82.35%. The IL-based UAE is free of volatile organic solvents, and consumes less sample, time and solvent, compared with regular ultrasonic and Soxhlet extraction. There was no obvious difference in the extraction yields of active constitutions obtained by the three extraction methods.

Topics: Boraginaceae; Chemical Fractionation; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Ionic Liquids; Linear Models; Naphthoquinones; Reproducibility of Results; Sensitivity and Specificity; Sonication

We recently discovered that 5, 8-O-dimethyl acylshikonin derivatives displayed the selectivity towards MCF-7 and no toxicity to normal cells. Herein, a series of the corresponding 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives were synthesized starting from shikonin. In vitro evidence of the cytotoxicities indicated that most of thecompounds were more active than or comparative to shikonin and retained the selectivity against MCF-7, MDA-MB-231 besides no toxicity in the normal cells. Also, in vivo anticancer activity of the positional isomers 5p, 6c further showed that 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives were more active than their corresponding 2-isomers. Thus, we may conclude that the position of the side chain of shikonin attached to 5,8-dimethoxy -1,4-naphthoquinone is associated with the antitumor activity.

Topics: Acylation; Animals; Antineoplastic Agents; Cell Survival; Drug Screening Assays, Antitumor; Female; Fibroblasts; Humans; Inhibitory Concentration 50; Isomerism; Male; Mice; Naphthoquinones; Neoplasms; Structure-Activity Relationship; Tumor Cells, Cultured

In pursuit of strong shikalkin-producing cell lines, seeds of the Iranian Arnebia euchroma were collected from Dena altitudes in the central Zagross. Chemical analysis showed that the dried root of the plant contained about 8.5% (w/w) shikalkin pigment. The root explants of the young plantlets, obtained from the germinated seeds, were used for establishing callus. Then, parameters effective on proliferation and pigment production of the resulting calli were studied in detail. Accordingly, two modified media called mLS and mM9 were optimized for propagation and pigment production, respectively. Using these media, the biomass of the A. euchroma calli was increased to 600%, and the pigment production reached to a maximum of 16.3  mg per gram of the wet biomass in a period of a subculture (21 days). Parallel to these experiments, the antimicrobial activity of shikalkin pigment was examined on some fungi and gram-positive and gram-negative bacteria. Results indicated that the pigment was almost ineffective on fungi and gram-negative bacteria, but it was meaningfully effective against Micrococcus luteus.

Topics: Anti-Bacterial Agents; Bacteria; Boraginaceae; Fungi; Naphthoquinones; Plant Extracts; Plant Roots; Seeds; Tissue Culture Techniques

A series of naphthoquinones was tested for activity against both extracellular promastigote and intracellular amastigote Leishmania major GFP in vitro. In parallel, the compounds were evaluated for cytotoxic effects against bone marrow-derived macrophages (BMM Φ) as a mammalian host cell control. Most of the compounds noticeably inhibited the growth of extracellular parasites (IC (50) 0.5 to 6 µM) and the intracellular survival of L. major GFP amastigotes (IC (50) 1 to 7 µM) when compared with the antileishmanial drug amphotericin B (IC (50) of 2.5 and 0.2 µM, respectively). In general, antiprotozoal activity and host cell cytotoxicity seemed to increase in parallel. Conspicuously, the cytotoxic effect was less pronounced on infected host cells when compared with that on noninfected cells. Concerning structure/activity relationships for the tested naphthoquinones, some interesting structural features emerged from this study. Introduction of a methyl or methoxyl group at C-2 of the parent 1,4-naphthoquinone slightly increased the antileishmanial activity against clinically relevant amastigotes, while the presence of a hydroxyl function in this position dramatically reduced the effectiveness. In contrast, hydroxylation at C-5 and dihydroxy substitution at C-5 and C-8 significantly enhanced the antiprotozoal activity. Similarly, the presence of a side chain hydroxyl group PERI to a carbonyl function as represented in the series of shikonin/alkannin derivatives increased the activity when compared with substituted analogs. Within the series of naphthoquinones tested, the dimeric mixture of vaforhizin and isovaforhizin showed the highest activity IN VITRO against the clinically relevant intracellular amastigote with an IC (50) of 1.1 µM. With IC (50) values mostly in the range of 1-3 µM, the shikonin/alkannin derivatives proved to be similarly considerably leishmanicidal. None of the compounds tested was capable to induce NO production known to play a crucial role in the host resistance against intracellular pathogens, excluding activation of microbicidal mechanisms in macrophages. The mode of action apparently depended on the substitution pattern, associated with the electrophilicity of the naphthoquinone or the efficiency of redox cycling. Conspicuously, members oxygenated in the quinone ring proved to be leishmanicidal when coincubated with glutathione, while the majority of the remaining compounds lost activity.

Topics: Amphotericin B; Animals; Antiprotozoal Agents; Bignoniaceae; Boraginaceae; Culture Media; Drosera; Flow Cytometry; Glutathione; Green Fluorescent Proteins; Hydroxylation; Inhibitory Concentration 50; Leishmania; Macrophages; Mammals; Naphthoquinones; Organisms, Genetically Modified; Parasitic Sensitivity Tests; Structure-Activity Relationship

To investigate the effects of shikonin on phorbol myristate acetate (PMA) plus cyclic adenosine monophosphate (cAMP)-induced T helper (T(H)) 2 cell cytokine production, and the underlying mechanism.. We used activated EL-4 murine T-lymphoma cells, which produce interleukin (IL)-4 and IL-5, but not interferon (IFN)-γ, as T(H)2 cell-like cells and treated them with PMA+cAMP to investigate the effects of shikonin on T(H)2 cytokines, transcriptional factors, and the related mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway.. The data show that shikonin inhibited the PMA+cAMP-induced mRNA and protein expression of IL-4 and IL-5 via the downregulation of GATA-binding protein-3 (GATA-3) and c-musculoaponeurotic fibrosarcoma (Maf) but not T-box expressed in T cells (T-bet). Moreover, shikonin suppressed the phosphorylation of p38, inhibitor of κB (IκB) kinase (IKK)-β and IκB-α, and the subsequent IκB-α degradation induced by PMA+cAMP; however, the PMA+cAMP-induced phosphorylation of extracellular signal-related kinase (ERK), which resulted in minor inhibition and phosphorylation of c-Jun N-terminal kinase (JNK), seemed to be unaffected by shikonin treatment.. This study suggests that downregulation of GATA-3 and c-Maf via the suppression of p38, IKK-β and IκB-α phosphorylation might contribute to the inhibitory effect of shikonin on mitogen-induced IL-4 and IL-5 production in EL-4T cells. Furthermore, shikonin is a potential drug for treating allergic diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Cell Line, Tumor; Cytokines; Down-Regulation; Drugs, Chinese Herbal; GATA3 Transcription Factor; I-kappa B Kinase; I-kappa B Proteins; Interleukin-4; Interleukin-5; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogens; Naphthoquinones; Proto-Oncogene Proteins c-maf; T-Lymphocytes

By studying on the factors influencing the dynamic changes of shikonin and its derivant,to interpret the great loss of these components in concentrating process of percolate of Arnerbia euchroma.. By sampling dynamically,and using HPLC for detection, dynamic changes of 5 components of shikonin and its derivant and relative factors were investigated during concentrating process at 50 degrees C, and influence of three key factors including temperature,pH and ethanol content on these components' stability were further investigated through single factor and multiple factors tests.. The content of 5 major components lost greatly during concentrating process, with volume and ethanol content decrease continually and pH from 5.56 to 4.5. Single factor and multiple factors tests about three key factors including temperature, pH and ethanol content indicated that these 5 components remained stable for 6 hours under condition of 40-60 degrees C, pH 3-7 and ethanol content 35%-70%. However,these components will precipitatie when the ethanol content of percolate became very low, which led to much lower content results and "loss phenomenon" of shikonin and its derivant.. The true causes for "great loss" of shikonin and its derivant during concentrating process do not lie in their chemical transformation due to poor stability, but lie in continual precipitation of these liposoluble components during concentrating process, which leads to content decrease significantly.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Drug Stability; Ethanol; Hydrogen-Ion Concentration; Naphthoquinones; Plant Roots; Plants, Medicinal; Technology, Pharmaceutical; Temperature; Time Factors

There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells.. Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally) once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin), plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium.. Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Blood Glucose; Calcium; Cell Line; Diabetes Mellitus, Experimental; Glucose Transporter Type 4; Insulin; Male; Muscle Cells; Muscle Fibers, Skeletal; Muscle, Skeletal; Naphthoquinones; Oxygen Consumption; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-akt; Rats

The objective of this study was to screen for novel quorum-sensing inhibitors (QSIs) from traditional Chinese medicines (TCMs) that inhibit bacterial biofilm formation. Six of 46 active components found in TCMs were identified as putative QSIs based on molecular docking studies. Of these, three compounds inhibited biofilm formation by Pseudomonas aeruginosa and Stenotrophomonas maltophilia at a concentration of 200 µM. A fourth compound (emodin) significantly inhibited biofilm formation at 20 µM and induced proteolysis of the quorum-sensing signal receptor TraR in Escherichia coli at a concentration of 3-30 mM. Emodin also increased the activity of ampicillin against P. aeruginosa. Therefore, emodin might be suitable for development into an antivirulence and antibacterial agent.

Topics: Anthraquinones; Anti-Bacterial Agents; Biofilms; Coumarins; Drugs, Chinese Herbal; Emodin; Escherichia coli; Microbial Sensitivity Tests; Naphthoquinones; Pseudomonas aeruginosa; Quorum Sensing; Stenotrophomonas maltophilia

Glutathione-S-transferase (GST) is a cytoplasmic protein responsible for detoxification, but the effect of the enzyme on cell biological events, including proliferation and migration, has never been reported. Thus, we evaluated the detoxification effect of in vitro-applied GST on cancer cell proliferation and migration. Assays for proliferation and migration of human breast cancer cells in the presence of GST were carried out. Binding of GST on the surface of the cancer cells was studied by flow cytometry. Detoxification through GST pathway was studied in the presence of shikonin. The effective dosage of GST in enhancement of cell proliferation was 10-50 nM, and the cell migration could be significantly enhanced after 6 hours in the presence of 2-50 nM GST. Therefore, overall cell proliferation and migration could be enhanced in the presence of 10nM or greater concentration of GST, and 15 μM shikonin-induced toxification of the cancer cells could be neutralized by 1.0 μM GST. Flow cytometry showed that GST directly bound to the surface of the cancer cells, and this was confirmed by fluorescence confocal microscopic observation. It is concluded that human class π-GST enhances proliferation and migration of human breast cancer cells by means of direct binding to the cell surface and maintaining cell viability by detoxification.

Topics: Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Cell Survival; Female; Flow Cytometry; Glutathione Transferase; Humans; Metabolic Detoxication, Phase II; Microscopy, Confocal; Naphthoquinones; Protein Binding; Recombinant Proteins

Although shikonin, a naphthoquinone derivative, has showed anti-cancer activity, its precise molecular anti-tumor mechanism remains to be elucidated. In this study, we investigated the effects of shikonin on human hepatocellular carcinoma (HCC) in vitro and in vivo. Our results showed that shikonin induced apoptosis of Huh7 and BEL7402 but not nontumorigenic cells. ROS generation was detected, and ROS scavengers completely inhibited shikonin-induced apoptosis, indicating that ROS play an essential role. Although the JNK activity was significantly elevated after shikonin treatment, JNK was not linked to apoptosis. However, downregulation of Akt and RIP1/NF-κB activity was found to be involved in shikonin-induced apoptosis. Ectopic expression of Akt or RIP1 partly abrogated the effects of shikonin, and Akt inhibitor and RIP1 inhibitor synergistically induced apoptosis in conjunction with shikonin treatment. ROS scavengers blocked shikonin-induced inactivation of Akt and RIP1/NF-κB, but Akt or RIP1/NF-κB did not regulate ROS generation, suggesting that Akt and RIP1/NF-κB signals are downstream of ROS generation. In addition, the results of xenograft experiments in mice were consistent with in vitro studies. Taken together, our data show that shikonin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in HCC cells through the ROS/Akt and RIP1/NF-κB pathways.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Naphthoquinones; Reactive Oxygen Species; Structure-Activity Relationship; Tumor Cells, Cultured

To discover reasons for great loss of shikonin during the concentrating process of percolate of Arnerbia euchroma (Royle) Johnst and develop a reasonable method for determination of shikonin.. Shikonin was selected as index, optimized chromatographic condition was used for analyzing the affection of alcohol content and crude drug content of sample solution on determination of shikonin, furthermore, reasonable sample preparation and determination methodology was examined and defined.. The optimized chromatographic condition was as follows: shikonin was analyzed with a Zorbax Extend C-18 column (4.6 mm x 250 mm, 5 microm), methanol: water (82: 18) as the mobile phase, the column was maintained at 35 degrees C, the flow rate was 1.0 mL min(-1), detection wavelength was set at 516 nm and the time for analysis reduced from 40 min to 24 min. Alcohol content of sample solution influenced determination results significantly, peak area of equal content shikonin in low alcohol content (<40%) was only about 20% - 30% of that of high alcohol content (>70%), the reasonable content of sample solution were 0.0167 - 0.083 g mL(-1) with alcohol content above 40%. The method showed good linearity, precision, reproducibility and accuracy.. The alcohol content of sample solution correlated with peak area closely for the first time, which indicate another important reason for "great loss" of shikonin during concentration process is that too much low ethanol content in test solution leads too much low results. The new method can detect shikonin more effectively and more reasonably and can monitor production process with high efficiency and low cost.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Drug Stability; Drugs, Chinese Herbal; Ethanol; Naphthoquinones; Plant Roots; Plants, Medicinal; Reproducibility of Results; Solvents; Technology, Pharmaceutical

Shikonin, 5,6-dihydroxyflavone-7-glucuronic acid, is the main ingredient of Lithospermum erythrorhizon Sieb. et Zucc, and was reported to have various biological activities including antiinflammatory, anticancer, antimicrobial and others. This study aimed to elucidate, for the first time, the antiobesity activity of shikonin and its mechanism of action. Shikonin was found to inhibit fat droplet formation and triglyceride accumulation in 3T3-L1 adipocytes. The half inhibitory concentration, IC(50), for the inhibition of triglyceride accumulation was found to be 1.1 microM. The expression of genes involved in lipid metabolism, such as FABP4 and LPL, were significantly inhibited following shikonin treatment. Shikonin also inhibited the ability of PPAR gamma and C/EBP alpha, the major transcription factors of adipogenesis, to bind to their target DNA sequences. The expressions of mRNA and protein of PPAR gamma and C/EBPa were significantly down-regulated following shikonin treatment. Among the upstream regulators of adipogenesis, only SREBP1C was found to be down-regulated by shikonin. The results of this study suggest that shikonin down-regulates the expression of SREBP1C and subsequently the expression of PPAR gamma and C/EBP alpha. Together, these changes result in the down-regulation of lipid metabolizing enzymes and reduced fat accumulation.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Animals; Anti-Obesity Agents; CCAAT-Enhancer-Binding Protein-alpha; Gene Expression Regulation; Inhibitory Concentration 50; Lipid Metabolism; Mice; Naphthoquinones; PPAR gamma; RNA, Messenger; Sterol Regulatory Element Binding Protein 1; Triglycerides

Shikonin (SK) has been isolated and identified as a key bioactive component in an herbal plant, Shikon (gromwell). In this study, we investigated antiestrogen activity of SK in breast cancer cells. In human breast cancer cells, we observed that treatment with SK inhibits tumor cell growth in estrogen receptor alpha (ERalpha)-positive, but not ERalpha-negative breast cancer cells. Estrogen-dependent cell growth was inhibited by co-treatment with SK. A potential molecular mechanism by which SK inhibits estrogen action was explored. We found that SK has no effect on ERalpha mRNA expression, but decreases its protein level. This effect is associated with an increase in ubiquitinated ERalpha for degradation. Our results suggest that SK downregulates ERalpha protein through a proteasome-mediated pathway. We also found that the treatment with SK inhibits estrogen-induced estrogen response elements reporter gene activity. Furthermore, SK inhibits recruitment of ERalpha at the estrogen-dependent gene promoters, and subsequently suppresses gene expression. Finally, co-treatment with SK enhanced sensitivity of breast cancer cells to endocrine therapy. Collectively, our studies suggested that SK has a potential for antihormone therapy in ERalpha-positive breast cancer cells, and should serve as a target for new drug developments.

Topics: Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; Estrogen Receptor alpha; Estrogen Receptor Modulators; Female; Gene Expression; Humans; Immunoblotting; Naphthoquinones; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD'(2) value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation-response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 microM. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC(50)s, 4 - 7 microM), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.

Topics: Animals; Aorta, Thoracic; Cell Line; Dose-Response Relationship, Drug; Macrophages; Male; Mice; Naphthoquinones; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Wistar; Vasodilation

To investigate the role of NF-kappaB signal transduction pathway in apoptosis induced by shikonin in human tongue squamous cell carcinoma Tca-8113 cell line.. Expression of IkappaBa, phosphatase-IkappaBa, bcl-2 and Bax proteins were detected by Western blot, NF-kappaB DNA-binding activity was detected by electrophoretic mobility shift analysis (EMSA), and activities of caspase 3, caspase 8 and caspase 9 were analyzed by enzyme linked immunosorbent assay(ELISA). The data was analyzed by one-way ANOVA test and t test using SPSS12.0 software package.. The expression of phosphatase-IkappaBa protein and the nuclear NF-kappaB DNA-binding activity was significantly decreased in shikonin treated cells by Western and EMSA. Bcl-2 protein expression was also decreased in the process. The activity of all the three proteases was elevated and pancaspase inhibitor Z-Asp-CH2-DCB could protect Tca8113 cells from shikonin-induced apoptosis(P=0.02).. Anti-tumor effects of shikonin in Tca-8113 cells act at least partially through the inactivation of NF-kappaB pathway and subsequent activation of protease caspase family. Pharmacologic inhibition of the NF-kappaB activity by shikonin might be a powerful treatment option for OSCC.

Topics: Apoptosis; Aspartic Acid; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Humans; Mouth Neoplasms; Naphthoquinones; NF-kappa B; Signal Transduction

Eleven shikonin glycosides were synthesized and evaluated for their antitumor activity in vitro. Some of them were found to exhibit cytotoxic activities against both drug sensitive cell lines (K562, MCF-7 and HL60) and their drug resistant cell sublines (K562/ADR, MCF-7/ADR and HL60/ADR).

Topics: Antineoplastic Agents; Glycosides; Humans; Inhibitory Concentration 50; Naphthoquinones

In the present paper we studied the effect of shikonin on ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and determined the mechanisms through which shikonin might exert its topical anti-inflammatory action.. Acute ear oedema was induced in mice by topical application of TPA. The in vitro assays used macrophages RAW 264.7 cells stimulated with lipopolysaccharide. Cyclooxygenase-2, inducible nitric oxide synthase, protein kinase Calpha, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (pERK), c-Jun N-terminal kinase (JNK), pJNK, p38, p-p38, p65, p-p65, inhibitor protein of nuclear factor-kappaB (NF-kappaB) (IkappaBalpha) and pIkappaBalpha were measured by Western blotting, activation and binding of NF-kappaB to DNA was detected by reporter gene and electrophoretic mobility shift assay, respectively, and NF-kappaB p65 localization was detected by immunocytochemistry.. Shikonin reduced the oedema (inhibitory dose 50 = 1.0 mg per ear), the expression of cyclooxygenase-2 (70%) and of inducible nitric oxide synthase (100%) in vivo. It significantly decreased TPA-induced translocation of protein kinase Calpha, the phosphorylation and activation of ERK, the nuclear translocation of NF-kappaB and the TPA-induced NF-kappaB-DNA-binding activity in mouse skin. Moreover, in RAW 264.7 cells, shikonin significantly inhibited the binding of NF-kappaB to DNA in a dose-dependent manner and the nuclear translocation of p65.. Shikonin exerted its topical anti-inflammatory action by interfering with the degradation of IkappaBalpha, thus inhibiting the activation of NF-kappaB.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Nucleus; Disease Models, Animal; Dose-Response Relationship, Drug; Edema; Female; I-kappa B Proteins; Inflammation; Inhibitory Concentration 50; Macrophages; Mice; Naphthoquinones; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Transport; Tetradecanoylphorbol Acetate

Isohexenylnaphthazarins (IHN), commonly known as alkannins and shikonins (A/S), are potent pharmaceutical substances with a wide spectrum of wound healing, antimicrobial, anti-inflammatory, and antitumor activity. Purification of A/S is crucial for their use in pharmaceuticals and for biological experimentation. Dimeric and oligomeric A/S derivatives co-exist with the active monomeric ones in most of the samples produced either by (semi)-synthesis or biotechnologically or isolated from natural products. Oligomeric A/S derivatives have not been studied for biological activity hitherto and a method to isolate them is essential.In the present study, solid-phase extraction (SPE) was applied for purification of commercial samples and isolation of monomeric and oligomeric A/S fractions, testing several stationary phases. Sephadex LH-20 cartridges achieved efficient purification for commercial samples containing both monomeric and dimeric A/S derivatives and also separation and isolation of both pure monomeric and dimeric A/S fractions for biological experiments. A high-performance liquid chromatography-diode array detection method was applied for detection, identification and quantification of monomeric and oligomeric shikonin fractions.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Chromatography, Liquid; Dextrans; Naphthoquinones; Solid Phase Extraction

Shikonin, a major ingredient in the Chinese traditional herb Lithospermum erythrorhixon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we delineated the molecular mechanisms of shikonin in the apoptosis of 143B osteosarcoma cells. Shikonin reduced the cell viability of 143B cells in a dose- and time-dependent manner. The IC(50) at 24 h and 48 h for 143B cells was 4.55 and 2.01microM, respectively. A significantly elicited hypodiploid cell population was found in cells treated with 2, 4, and 8microM shikonin for 24 h. Moreover, treatment with shikonin induced reactive oxygen species (ROS) generation, increased extracellular signal-regulated kinase (ERK) phosphorylation, decreased B-cell lymphoma-2 (Bcl2) expression, and was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage. Pretreatment with the antioxidant agent N-acetyl cysteine (NAC) not only reversed shikonin-induced ROS generation but also significantly attenuated the cytotoxic effects of shikonin in 143B cells. Furthermore, NAC attenuated shikonin-induced ERK phosphorylation. Taken together, our results reveal that shikonin increased ROS generation and ERK activation, and reduced Bcl2, which consequently caused the cells to undergo apoptosis. Therefore, shikonin may be a promising chemotherapeutic agent for osteosarcoma treatment.

Topics: Acetylcysteine; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Extracellular Signal-Regulated MAP Kinases; Humans; Inhibitory Concentration 50; Naphthoquinones; Osteosarcoma; Phosphorylation; Phytotherapy; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

The aim of our study was to investigate the neuroprotective properties of shikonin, a naphthoquinone pigment isolated from the roots of the traditional Chinese herb Lithospermum erythrorhizon. In the present study, mice were divided randomly into sham, model, shikonin and edaravone-treated groups. Shikonin (50, 25, and 12.5mg/kg, i.g.) or maize oil was administered three times before ischemia and once at 2h after the onset of ischemia. Mice were anesthetized with chloral hydrate and subjected to middle cerebral artery 2h of occlusion and then 22h of reperfusion. Different antioxidant assays were employed in order to evaluate the antioxidant activities of shikonin in vitro. Neurological deficit, infarct size, histopathology changes and oxidative stress markers were evaluated after 22h of reperfusion. In comparison with the model group, treatment with shikonin significantly decreased neurological deficit scores, infarct size, the levels of malondialdehyde(MDA), carbonyl and reactive oxygen species, and attenuated neuronal damage, up-regulated superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) activities and reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Taken together, these results suggested that the neuroprotective effects of shikonin against cerebral ischemia/reperfusion injury may be attributed to its antioxidant effects.

Topics: Animals; Antioxidants; Biomarkers; Brain Ischemia; Dose-Response Relationship, Drug; Glutathione; Infarction, Middle Cerebral Artery; Male; Malondialdehyde; Mice; Mitochondria; Naphthoquinones; Neuroprotective Agents; Oxidative Stress; Oxidoreductases; Protein Carbonylation; Random Allocation; Reactive Oxygen Species; Reperfusion Injury; Up-Regulation

Exposure to higher levels of estrogen produces genotoxic metabolites that can stimulate mammary tumorigenesis. Induction of NF-E2-related factor 2 (NRF2)-dependent detoxifying enzymes (e.g., NAD(P)H-quinone oxidoreductase 1 (NQO1)) is considered an important mechanism of protection against estrogen-associated carcinogenesis because they would facilitate removal of toxic estrogens. Here, we studied the impact of estrogen-receptor (ER) signaling on NRF2-dependent gene transcription. In luciferase assay experiments using the 5-flanking region of the human NQO1 gene promoter, we observe that ERα ligand-binding domain (LBD) is required for estrogen inhibition of NQO1 promoter activity in estrogen-dependent breast cancer cells. Chromatin immunoprecipitation (ChIP) assay shows that estrogen recruits ERα and a class III histone deacetylase SIRT1 at the NQO1 promoter, leading to inhibition of NQO1 transcription. Inhibition of ERα expression by the antiestrogen shikonin reverses the inhibitory effect of estrogen on NQO1 expression. As a consequence, a chemoprevention study was undertaken to monitor the impact of shikonin on DNA lesions and tumor growth. Treatment of MCF-7 breast cancer cells with shikonin inhibits estrogen-induced 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA damage. NQO1 deficiency promotes estrogen-dependent tumor formation, and shikonin inhibits estrogen-dependent tumor growth in an NQO1-dependent manner in MCF-7 xenografts. These results suggest that estrogen-receptor signaling pathway has an inhibitory effect on NRF2-dependent enzymes. Moreover, shikonin reverses the inhibitory effects of estrogen on this pathway and may contribute to breast cancer prevention.

Topics: Animals; Antineoplastic Agents, Phytogenic; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; DNA Damage; Dose-Response Relationship, Drug; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; NF-E2-Related Factor 2; Ovariectomy; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Sirtuin 1; Transcription, Genetic; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Shikonin exhibits a wide range of anti-inflammatory actions. Here, we assessed its effects on maturation of murine bone marrow-derived dendritic cells (BM-DCs) and on allergic reactions in a murine model of asthma.. Cultured murine BM-DCs were used to investigate the effects of shikonin on expression of cell surface markers and their stimulation of T-cell proliferation and cytokine production. The therapeutic potential of shikonin was evaluated in a model of allergic airway disease.. Shikonin dose-dependently inhibited expression of major histocompatibility complex class II, CD80, CD86, CCR7 and OX40L on BM-DCs, induced by a mixture of ovalbumin (OVA; 100µg·mL(-1) ) and thymic stromal lymphopoietin (TSLP; 20ng·mL(-1) ). Shikonin-treated BM-DCs were poor stimulators of CD4(+) T lymphocyte and induced lower levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor (TNF)-α release by responding T-cells. After intratracheal instillation of shikonin in OVA-immunized mice, OVA challenge induced lower IL-4, IL-5, IL-13, TNF-α and eotaxin release in bronchial alveolar lavage fluid, lower IL-4 and IL-5 production in lung cells and mediastinal lymph node cells and attenuated OVA-induced lung eosinophilia and airway hyperresponsiveness.. Shikonin effectively suppressed OVA + TSLP-induced BM-DC maturation in vitro and inhibited allergic inflammation and airway hyperresponsiveness in a murine model of asthma, showing good potential as a treatment for allergic asthma. Also, our model provides a novel platform for screening drugs for allergic diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Asthma; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; Cytokines; Dendritic Cells; Drugs, Chinese Herbal; Female; Inflammation; Interleukin-13; Interleukin-4; Interleukin-5; Lung; Mice; Mice, Inbred BALB C; Naphthoquinones; Ovalbumin; Thymic Stromal Lymphopoietin; Tumor Necrosis Factor-alpha

Functional comparative genomic analysis of the cellular immunological effects of different anti-inflammatory phytocompounds is considered as a helpful approach to distinguish the complex and specific bioactivities of candidate phytomedicines. Using LPS-stimulated THP-1 monocytes, we characterize here the immunomodulatory activities of three single phytocompounds (emodin, shikonin, and cytopiloyne) and a defined phytocompound mixture extracted from Echinacea plant (BF/S+L/Ep) by focused DNA microarray analysis of selected immune-related genes.. Shikonin and emodin significantly inhibited the early expression (within 0.5 h) of approximately 50 genes, notably cytokines TNF-α, IL-1β and IL-4, chemokines CCL4 and CCL8, and inflammatory modulators NFATC3 and PTGS2. In contrast, neither cytopiloyne nor BF/S+L/Ep inhibited the early expression of these 50 genes, but rather inhibited most late-stage expression (~12 h) of another immune gene subset. TRANSPATH database key node analysis identified the extracellular signal-regulated kinase (ERK) 1/2 activation pathway as the putative target of BF/S+L/Ep and cytopiloyne. Western blot confirmed that delayed inactivation of the ERK pathway was indeed demonstrable for these two preparations during the mid-stage (1 to 4 h) of LPS stimulation. We further identified ubiquitin pathway regulators, E6-AP and Rad23A, as possible key regulators for emodin and shikonin, respectively.. The current focused DNA microarray approach rapidly identified important subgenomic differences in the pattern of immune cell-related gene expression in response to specific anti-inflammatory phytocompounds. These molecular targets and deduced networks may be employed as a guide for classifying, monitoring and manipulating the molecular and immunological specificities of different anti-inflammatory phytocompounds in key immune cell systems and for potential pharmacological application.

Topics: Anti-Inflammatory Agents; Cell Death; Cell Line; Cluster Analysis; Emodin; Enzyme Activation; Gene Expression Profiling; Gene Expression Regulation; Genes, Switch; Genomics; Glucosides; Humans; Immunity; Kinetics; Lipopolysaccharides; MAP Kinase Signaling System; Monocytes; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Plant Extracts; Polyynes; Signal Transduction

A set of twenty-two 5,8-O-dimethyl acylshikonin derivatives were designed and synthesized starting from shikonin. The cell-based investigation demonstrated that these dimethylated derivatives were less active than or equally effective to shikonin. However, the selective cytotoxicities toward MCF-7 were found among these derivatives, together with no toxicity in the normal cell. Furthermore, compounds 3f, 3p, 3r were subjected to KM mice suffering from S-180 carcinoma subcutaneously, which possessed more potent than Fluorouracil, a typical anticancer drug used clinically. So we may conclude that the modification to the mother nucleus of shikonin via the methylation is an available approach to acquiring anti-tumor agents with higher selectivity and lower toxicity.

Topics: Antineoplastic Agents; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Naphthoquinones; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

Geranyl pyrophosphate (GPP) and p-hydroxybenzoate (PHB) are the basic precursors involved in shikonins biosynthesis. GPP is derived from mevalonate (MVA) and/or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway(s), depending upon the metabolite and the plant system under consideration. PHB, however, is synthesized by only phenylpropanoid (PP) pathway. GPP and PHB are central moieties to yield shikonins through the synthesis of m-geranyl-p-hydroxybenzoate (GHB). Enzyme p-hydroxybenzoate-m-geranyltransferase (PGT) catalyses the coupling of GPP and PHB to yield GHB. The present research was carried out in shikonins yielding plant arnebia [Arnebia euchroma (Royle) Johnston], wherein no molecular work has been reported so far. The objective of the work was to identify the preferred GPP synthesizing pathway for shikonins biosynthesis, and to determine the regulatory genes involved in the biosynthesis of GPP, PHB and GHB.. A cell suspension culture-based, low and high shikonins production systems were developed to facilitate pathway identification and finding the regulatory gene. Studies with mevinolin and fosmidomycin, inhibitors of MVA and MEP pathway, respectively suggested MVA as a preferred route of GPP supply for shikonins biosynthesis in arnebia. Accordingly, genes of MVA pathway (eight genes), PP pathway (three genes), and GHB biosynthesis were cloned. Expression studies showed down-regulation of all the genes in response to mevinolin treatment, whereas gene expression was not influenced by fosmidomycin. Expression of all the twelve genes vis-à-vis shikonins content in low and high shikonins production system, over a period of twelve days at frequent intervals, identified critical genes of shikonins biosynthesis in arnebia.. A positive correlation between shikonins content and expression of 3-hydroxy-3-methylglutaryl-CoA reductase (AeHMGR) and AePGT suggested critical role played by these genes in shikonins biosynthesis. Higher expression of genes of PP pathway was a general feature for higher shikonins biosynthesis.

Topics: Boraginaceae; Gene Expression Regulation, Plant; Genes, Plant; Geranyltranstransferase; Hydroxymethylglutaryl CoA Reductases; Naphthoquinones; Parabens; Polyisoprenyl Phosphates

To develop a HPLC method for determination of matrine, oxymatrine; ferulic acid, L-shikonin and beta, beta-dimethylacrylshikonin in compound preparatioti of Dangguishuan.. The chromatogtaphic separation was performed on a Kromasil ODS C18 column (4.6 mm x 250 mm, 5 microm) maintaining at 30 degrees C during the whole process. The mobile phase consisted of methanol and 0.1% triethylamine aqueous solution (adjusted with phosphoric acid, pH 3) at a flow rate of 1.0 mL x min(-1). The detection wavelength was set at 220 nm for matririne and oxymatrine, 316 nm for ferulic acid, 516 nm for L-shikonin and beta, beta-dimethylacrylshikonin, respectively.. All the compounds showed good linearity (r > 0.9996) in the range of the test concentrations, and the average recoveries of the method is betwuen 96.92% and 102.22%, RSD < 3.1%.. The method is proved to be credible, sensitive, accurate and repeatable. It can be applied to determine of matrine, oxymatrine, ferulic acid, L-shikonin and beta, beta-dimethylacrylshikonin in compound preparation of Dangguishuan simultaneously, and provide a basal method of quality control to this preparation and other relative preparations.

Topics: Alkaloids; Chromatography, High Pressure Liquid; Coumaric Acids; Drugs, Chinese Herbal; Matrines; Naphthoquinones; Quinolizines

This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential (ΔΨm) disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in ΔΨm, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. Furthermore, we show that shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 h treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Growth Processes; Cell Line, Tumor; Dose-Response Relationship, Drug; fas Receptor; Hep G2 Cells; Humans; Isoflavones; Liver Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Electron; Mitochondria; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; S Phase; Signal Transduction

To investigate the anti-inflammatory or immunomodulatory effect of shikonin on early stage and established murine collagen-induced arthritis (CIA).. Mouse were injected intraperitoneally with shikonin (5 mg/kg) for 10 days along before or after the onset of CIA. The arthritis response was monitored visually by macroscopic scoring. Reverse transcription-polymerase chain reaction and western blotting were employed to determine the mRNA and protein expression of cytokine in patella with adjacent synovium in CIA mouse. Histology of knee was used to assess the occurrence of cartilage destruction and bone erosion.. Shikonin (5 mg/kg) treatment along had no effect on macroscopic score and incidence of arthritis on early stage of CIA. However, a pronounced amelioration of macroscopic score and cartilage destruction was found in mouse treated with shikonin on established CIA for 10 days. Moreover, The mRNA levels of Th1 cytokines [tumor necrosis factor-alpha and interleukin (IL)-12] was significantly inhibited both in the synovial tissue and in the articular cartilage in treated groups compared with those in control groups, whereas the mRNA and protein levels of Th2 cytokines (IL-10 and IL-4) remained elevated throughout the treatment period. Moreover, the inflammatory cytokine, the mRNA and protein levels of IL-6 was down-regulated in mice with established CIA after treatment with shikonin. T-box expressed in T cells (T-bet) mRNA levels were decreased in shikonin compared with control group, and GATA-3 mRNA levels were higher than that in control group.. Shikonin treatment on established CIA can inhibit Th1 cytokines expression and induce Th2 cytokines expression in mice with established CIA. The inhibited effect of shikonin on Th1 cytokines expression may be mediated not only by inhibiting Th1 responses through T-bet mechanism, but also by inducing anti-inflammatory mediators such as IL-10 and IL-4 through a GATA-3 dependent mechanism.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Cartilage, Articular; Collagen Type II; Cytokines; GATA3 Transcription Factor; Gene Expression Regulation; Interleukins; Knee Joint; Mice; Naphthoquinones; RNA, Messenger; Synovial Membrane; Treatment Outcome

Alkannin and shikonin (A/S) and their derivatives have been found in the roots of several Boraginaceous species and are also produced through plant tissue cultures. The chiral compounds A/S are potent pharmaceutical substances with a wide spectrum of biological and pharmacological activities like wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant activity. High-speed counter-current chromatography (HSCCC) was applied for the first time to the separation, preparative isolation and purification of A/S and their esters from extracts of Alkanna tinctoria roots, as well as commercial samples. The constituents of HSCCC fractions and their purity were determined by high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS), since DAD cannot detect oligomeric A/S derivatives that are present in most of the samples containing the respective monomeric derivatives. The purity of HSCCC fractions was compared with the one of fractions isolated by column chromatography (CC) using as stationary phases silica gel and Sephadex LH-20. As shown, the purity of monomeric alkannin/shikonin was greater by HSCCC than CC separation of commercial A/S samples.

Topics: Boraginaceae; Chromatography; Chromatography, High Pressure Liquid; Mass Spectrometry; Naphthoquinones; Plant Extracts; Plant Roots

Endogenously occurring nitric oxide (NO) is involved in the regulation of shikonin formation in Onosma paniculatum cells. NO generated after cells were inoculated into shikonin production medium reached the highest level after 2 d of culture, which was 16 times that at the beginning of the experiment, and maintained a high level for 6 d. A nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor, sodium azide (SoA), consistent with their inhibition of NO biosynthesis, decreased shikonin formation significantly. This reduction could be alleviated or even abolished by exogenous NO supplied by sodium nitroprusside (SNP), suggesting that the inhibition of NO biosynthesis resulted in decreased shikonin formation. However, when endogenous NO biosynthesis was up-regulated by the elicitor from Rhizoctonia cerealis, shikonin production was enhanced further, showing a dependence on the elicitor-induced NO burst. Real-time PCR analysis showed that NO could significantly up-regulate the expression of PAL, PGT and HMGR, which encode key enzymes involved in shikonin biosynthesis. These results demonstrated that NO plays a critical role in shikonin formation in O. paniculatum cells.

Topics: Boraginaceae; Cells, Cultured; Culture Media; Electron Spin Resonance Spectroscopy; Gene Expression Regulation, Plant; Naphthoquinones; NG-Nitroarginine Methyl Ester; Nitric Oxide; RNA, Plant

We previously reported that shikonin could circumvent drug resistance mediated by P-gp, Bcl-2 and Bcl-xL, by induction of necroptosis. Here, we show that the naturally-occurring shikonin analogues (deoxyshikonin, acetylshikonin, isobutyrylshikonin, beta,beta-dimethylacrylshikonin, isovalerylshikonin, alpha-methyl-n-butylshikonin) could bypass drug resistance mediated by not only P-gp, Bcl-2, and Bcl-xL, but also two additional important drug-resistant factors MRP1 and BCRP1, by induction of necroptosis. The results strengthen the previous findings that necroptotic induction could circumvent a broad spectrum of cancer drug resistance.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; bcl-X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Drug Resistance, Neoplasm; Electrophoresis; Flow Cytometry; Humans; Microscopy, Electron; Multidrug Resistance-Associated Proteins; Naphthoquinones; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2

A series of shikonin analogues with side chain variants have been synthesized and evaluated for antitumor activity. These novel analogues show a broad spectrum of in vitro cytotoxicity against various cancer cell lines. Additionally, some analogues were also found to have the ability to decrease the expression level of HIF-1alpha in breast cancer cells MDA-MB-231 under hypoxia. The features of these analogues suggest their potential in cancer therapy.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Design; Drug Screening Assays, Antitumor; HeLa Cells; HL-60 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inhibitory Concentration 50; Models, Chemical; Naphthoquinones

Dysregulation of the ubiquitin-proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell-killing activity, and results from a clinical study using a shikonin-containing mixture demonstrated its safety and efficacy for the treatment of late-stage lung cancer. In this study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C(1) and C(4) of shikonin potentially interact with the catalytic site of beta 5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin-like activity of purified 20S proteasome (IC(50) 12.5 micromol/L) and tumor cellular 26S proteasome (IC(50) between 2-16 micromol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC-3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (I kappaB-alpha, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC-3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin and inhibition of the proteasome activity by shikonin contributes to its antitumor property.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Flow Cytometry; Humans; Male; Mice; Mice, Nude; Models, Molecular; Naphthoquinones; Neoplasms, Experimental; Protease Inhibitors; Proteasome Inhibitors; Survival Rate

Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Blotting, Western; Carcinoma, Lewis Lung; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Chickens; Chorioallantoic Membrane; Drugs, Chinese Herbal; Electrophoretic Mobility Shift Assay; Female; Immunoenzyme Techniques; Immunoprecipitation; Luciferases; Mice; Mice, Inbred C57BL; Naphthoquinones; Neovascularization, Pathologic; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Nine shikonin pigments: shikonin (S), acetylshikonin (AS), propionylshikonin (PS), isobutyrylshikonin (IBS), tiglylshikonin (TS), 3,3-dimethylacrylshikonin (DAS), angelylshikonin (ANS), 2-methyl-n-butyrylshikonin (MBS) and isovalerylshikonin (IVS) were identified in the root epidermis of Echium italicum L. for the first time. A new thin-layer chromatographic (TLC) method for the separation of enantiomers alkannin and shikonin proved only shikonin after saponification of the root extract, and was afterwards esterified with the corresponding acyl chloride to acquire seven standard compounds (all except ANS). The developed isocratic high-peformance liquid chromatographic (HPLC) methods with VIS and mass spectrometry (MS) detection, allowed for the first time simultaneous separation of all nine compounds with similar structures including positional and geometric isomers in a short time. Structures of the main five compounds (AS, IBS, ANS, MBS, IVS) isolated from the extract by a new semi-preparative HPLC on C18 have additionally been confirmed by (1)H and (13)C nuclear magnetic resonance spectra, which were reported for AS and MBS for the first time.

Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Echium; Mass Spectrometry; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Plant Roots

Degterev et al. previously demonstrated that death receptor mediated apoptosis could be diverted to necroptosis when apoptosis signaling was blocked, suggesting that necroptosis may function as a backup mechanism to insure the elimination of damaged cells under certain conditions when apoptosis was inhibited. Here, we show that shikonin-induced necroptosis can be reverted to apoptosis in the presence of necrostatin-1 (Nec-1), a specific necroptosis inhibitor and that the death mode switch is at least partially due to the conversion from mitochondrial inner membrane permeability to mitochondrial outer membrane permeability, which is associated with Bax translocation. The data combined with the previous reports support a notion that apoptosis and necroptosis may function as reciprocal backup mechanisms of cellular demise. To the best of our knowledge, this is the first study to document a conversion from necroptosis to apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Imidazoles; Indoles; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Naphthoquinones; Necrosis; Permeability

Steroid sulfatase (STS) has an important role in regulating the biosynthesis of estrogen within breast tumors. We aimed to investigate whether shikonin, an ingredient of Lithospermum erythrorhizon, could modulate STS expression in breast cancer cells. By MTT assay, shikonin inhibited the cell proliferation of breast cancer cells MCF-7 and SK-BR-3. Moreover, by semi-quantitative/quantitative reverse transcription polymerase chain reaction and dual-luciferase reporter based bioluminescent measurements, the mRNA and enzymatic activity levels of STS were decreased after shikonin treatment. Concluding, shikonin could act as a selective estrogen enzyme modulator by down-regulating the STS expression.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Enzymologic; Humans; Lithospermum; Naphthoquinones; RNA, Messenger; Steryl-Sulfatase

Novel electrospun poly(epsilon-caprolactone) (PCL)/poly(trimethylene carbonate) (PTMC) ultrafine composite fiber mats were prepared and used as drug-carrying materials to encapsulate the herbal medicine shikonin isolated from the plant Lithospermum erythrorhizon Sieb. et Zucc. The PCL/PTMC blended solutions in various ratios (9:1, 7:3, and 5:5, w/w) containing 1 and 5 wt.% shikonin were studied for electrospinning into nanoscale fiber mats. With good drug stability and high drug-loading efficacy, incorporation of shikonin in the polymer media did not appear to influence the morphology of the resulting fibers, as both the drug-free and the shikonin-loaded composite fibers remained unaltered, microscopically. The average diameter of the composite fibers decreased, and the morphology of the fibers became finer with the increasing content of PTMC. In vitro drug release studies demonstrated an initial rapid release of shikonin followed by a plateau after 11 h. It was found that the release behavior could be tailored by the PCL/PTMC blend ratio and drug-loading content. Moreover, the free radical scavenging activity and the antibacterial effects of the shikonin-loaded fiber mats indicated that it could act not only as a drug delivery system but also in the treatment of wound healing or dermal bacterial infections.

Topics: Anti-Bacterial Agents; Chemistry, Pharmaceutical; Dioxanes; Disk Diffusion Antimicrobial Tests; Dosage Forms; Drug Carriers; Drug Compounding; Drug Stability; Drugs, Chinese Herbal; Escherichia coli; Free Radical Scavengers; Kinetics; Nanofibers; Naphthoquinones; Particle Size; Polyesters; Polymers; Solubility; Staphylococcus aureus; Surface Properties; Technology, Pharmaceutical

A simple, precise, and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin (1) together with its derivatives acetylshikonin (2), and beta-acetoxyisovalerylshikonin (3) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP-18 F(254S )TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100-600 ng for shikonin and acetylshikonin, and 100-1800 ng for beta-acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1-3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively.

Topics: Anthraquinones; Boraginaceae; Chromatography, Thin Layer; Densitometry; Naphthoquinones; Species Specificity; Ultrasonics

The present study was performed to evaluate the potential protective effects of Shikonin extracted from Zicao on lupus nephritis (LN) using NZB/W F1 mice. Oral administration of Shikonin (24, 40 mg/kg body weight/d) or vehicle was applied to sixty female NZB/W F1 mice of 28-week-old with LN. Treatment with Shikonin for 14 weeks suppressed proteinuria dose-dependently with the mean proteinuria of 274.0 mg/dl and 160.3 mg/dl for low-dose and high-dose Shikonin groups, respectively, compared to 499.2 mg/dl for the vehicle. Also, Shikonin was observed to reduce circulating adhesion molecules significantly and down-regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression in kidney. However, anti-double stranded (ds)DNA antibody in mice with low or high Shikonin dose administration both exhibited no significant elevation, differing from vehicle group. Kidney histological examination showed that renal glomerular lesions were alleviated after Shikonin application. These results suggest that Shikonin has therapeutic effects on LN in NZB/W F1 mice, to which inhibition of anti-dsDNA may be potential contribution, and its part mechanism is related to suppression of mRNA expression of cell adhesion molecules (CAMs) in the kidney.

Topics: Animals; Boraginaceae; Cell Adhesion Molecules; Down-Regulation; Drugs, Chinese Herbal; Female; Kidney; Lithospermum; Lupus Nephritis; Mice; Mice, Inbred NZB; Naphthoquinones

A novel shikonin derivative, named bothriodumin was isolated from the alcoholic extract of the whole plant of Bothriospermum secundum Maxim. And its structure was characterized on the basis of spectroscopic methods such as 1D-, 2D-NMR and HR-ESIMS techniques.

Topics: Boraginaceae; Magnetic Resonance Spectroscopy; Naphthoquinones; Plant Extracts; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet

To investigate the effects of shikonin on the proliferation, expression of CXCR4 and the migratory responses to CXCL12 in colorectal carcinoma cell line SW480.. The proliferation of SW480 cells was assessed by MTT assay. Cell surface expression of CXCR4 was determined by flow cytometry. The migratory ability was determined by Transwell.. Shikonin inhibited the proliferation of SW480 cells in time- and concentration-dependent manner. The expression rate of CXCR4 in SW480 cells was 99.1%. After application of shikonin 0.01 micromol/L, 0.1 micromol/L and 1.0 micromol/L for 24 h, the expression rate of CXCR4 decreased to 76.0%, 59.1% and 35.5% respectively (F=1098.041, P <0.001), and the CXCL12-induced SW480 cell migratory inhibition rate was 25.2%, 38.5% and 55.7% respectively (F=48.970, P <0.001).. Besides having inhibiting tumor cell proliferation effect, Shikonin may also play a role in anti-metastasis via down-regulating the expression of CXCR4 and reducing the CXCL12-induced migratory response in colorectal carcinoma cell.

Topics: Cell Line, Tumor; Cell Proliferation; Chemokine CXCL12; Down-Regulation; Humans; Naphthoquinones; Receptors, CXCR4

Microglial cells are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. Shikonin, a naphthoquinone pigment from the root of Lithospermum erythrorhizon, has long been used as an ointment for wound healing in traditional oriental medicine. Shikonin has been reported to have antibacterial, antitumor, and anti-inflammatory effects. The aim of this study was to examine whether shikonin represses microglial activation. In a study of shikonin and five of its derivatives, isobutyrylshikonin (IBS) and isovalerylshikonin (IVS) were the most effective at inhibiting LPS-induced nitric oxide (NO) release from microglial cells. Reverse transcriptase real-time PCR analysis revealed that pretreatment of rat brain microglia with IBS and IVS attenuated the LPS-induced expression of mRNAs encoding inducible NO synthase, tumor necrosis factor (TNF)-alpha, interleukin-1beta, and cyclooxygenase-2. In rat brain microglia, IBS and IVS reduced the LPS-stimulated production of TNF-alpha and prostaglandin E2. In addition, IBS and IVS significantly decreased LPS-induced IkappaB-alpha phosphorylation and NF-kappaB DNA binding activity, as well as the phosphorylation of the ERK1/2 and Akt signaling proteins. In organotypic hippocampal slice cultures, propidium iodide staining revealed prominent cell death in the hippocampal layer after 72h of LPS treatment. Both IBS and IVS clearly blocked the effect of LPS on hippocampal cell death and inhibited LPS-induced NO production in culture medium. These results suggest that IBS and IVS provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Cell Survival; Cells, Cultured; Cerebral Cortex; Chromones; Dinoprostone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Microglia; Morpholines; Naphthoquinones; NF-kappa B; Nitrites; Oligonucleotides; Oncogene Protein v-akt; Polysaccharides; Rats; Time Factors; Tumor Necrosis Factor-alpha

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells (e.g., K562, LAMA84). Treatment of K562 cells with shikonin (e.g., 0.5 muM) resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species (ROS), striking activation of c-Jun-N-terminal kinase (JNK) and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events (i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis) following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cytochromes c; Fusion Proteins, bcr-abl; Humans; JNK Mitogen-Activated Protein Kinases; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitochondria; Naphthoquinones; Oxidative Stress; Proto-Oncogene Proteins c-abl; Proto-Oncogene Proteins c-bcr; Reactive Oxygen Species; Signal Transduction

Shikonin, a component of the herbal medicine "Shikon", is known to suppress inflammatory reactions, but its molecular targets are not identified. This study examines the effect of shikonin on human basophil degranulation response and aims to identify its targets.. Human basophils in isolated leukocytes from healthy volunteers' peripheral blood; recombinant human Syk and Lyn tyrosine kinases.. Histamine release from basophils stimulated with anti-IgE antibody was analyzed fluorimetrically. Syk and Lyn kinase activities were tested in Vitro with recombinant proteins and analyzed by off-chip mobility shift assay.. Shikonin dose-dependently inhibited the histamine release from basophils induced by anti-IgE antibody (IC50 = 2.6 +/- 1.0 microM; mean +/- SEM). A search for the target(s) of shikonin in the signal cascade of IgE-mediated activation showed that it strongly inhibits Syk (IC50 = 7.8 microM, in the recombinant kinase assay), which plays a pivotal role in the degranulation response. A less significant inhibition was found for Lyn, which phosphorylates FcepsilonRI-betagamma subunits and also Syk.. These results indicate that the inhibition of Syk-dependent phosphorylation events might underlie the blocked histamine release from human basophils, thus contributing to the anti-inflammatory effects of shikonin.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Basophils; Histamine Release; Humans; Immunoglobulin E; Intracellular Signaling Peptides and Proteins; Naphthoquinones; Protein-Tyrosine Kinases; Syk Kinase

Shikonin/alkannin (SA) derivatives, analogs of naphthoquinone pigments, are the major components of root extracts of the Chinese medicinal herb (Lithospermum erythrorhizon; LE) and widely distributed in several folk medicines. In the present study, the effect and the underline molecular mechanism of shikonin derivatives isolated from root extracts of Lithospermum euchroma on lipopolysaccharide (LPS)-induced inflammatory response were investigated.. Effects of five SA derivatives, including SA, acetylshikonin, beta,beta-dimethylacrylshikonin, 5,8-dihydroxy-1.4-naphthoquinone, and 1,4-naphthoquinone on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in mouse macrophage RAW264.7 cells were examined.. Data suggested that SA derivatives inhibited LPS-induced NO and PGE(2) production, and iNOS protein expression. RT-PCR analysis showed that SA derivatives diminished LPS-induced iNOS mRNA expression. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in LPS-stimulated RAW 264.7 cells was concentration-dependently suppressed by SA derivatives. SA inhibited NF-kappaB activation by prevention of the degradation of inhibitory factor-kappaB and p65 level in nuclear fractions induced by LPS.. Taken together, these results suggest that the anti-inflammatory properties of SA derivatives might result from inhibition of iNOS protein expression through the downregulation of NF-kappaB activation via suppression of phosphorylation of ERK, in LPS-stimulated RAW 264.7 cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Dinoprostone; Dose-Response Relationship, Drug; Down-Regulation; Lipopolysaccharides; Lithospermum; Macrophages; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction

Lithospermum erythrorhizon has been used for treatment of inflammatory diseases and cancer as a folk remedy. Based on the evidences that anti-inflammatory agents frequently exert antiangiogenic activity, thus we examined comparatively the antiangiogenic activities of three naphthoquinone derivatives (shikonin, acetylshikonin, and isobutyroylshikonin) isolated from the plant. Three derivatives exhibited weak cytotoxicity against human umbilical vein endothelial cells (HUVECs) with IC50 of over 20 microM. Shikonin had more specific inhibitory effects on proliferation and vascular endothelial growth factor (VEGF) production by VEGF compared with different derivatives. All of derivatives significantly suppressed the migration of VEGF treated HUVECs at different optimal concentrations. Also, shikonin and acetylshikonin significantly disrupted VEGF-induced tube formation. Furthermore, three derivatives effectively downregulated the expression of urokinase-type plasminogen activator (uPA), but not its receptor uPAR. Additionally, shikonin significantly inhibited tumor growth in LLC-bearing mice, whereas its derivatives had relatively mild effects. Taken together, our findings suggest that shikonin and its derivatives exhibit the antiangiogenic and antitumorigenic effects by suppressing proliferation and angiogenic factors.

Topics: Animals; Anthraquinones; Anti-Inflammatory Agents, Non-Steroidal; Carcinoma, Lewis Lung; Cell Movement; Cells, Cultured; Depression, Chemical; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Cells; Female; Humans; Lithospermum; Mice; Naphthoquinones; Neovascularization, Pathologic; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A

Beta,beta-dimethyl acryl shikonin is an extract from the root of plant Arnebia nobilis which has been shown to possess anti-cancer activity. However, its toxicity limited further development of shikonin as a therapeutic agent. Subsequently, several analogues of beta,beta-dimethyl acryl shikonin were synthesized. One of these analogues, shikonin 93/637 was found to be significantly less toxic compared to shikonin. This study is aimed to determine the cell cycle associated differences in the susceptibility of U937 cells to apoptosis induced by shikonin analogue 93/637 (SA). Lower concentrations of SA (approximately 100 nM) showed no significant changes in cell growth. However, higher concentrations (approximately 500 nM) resulted in growth inhibition of U937 cells after 48 h of treatment with SA as measured by MTT assay. Flow cytometric analysis showed that SA treatment resulted in blocking of cell cycle progression in G1 phase. Decreased expression of Cyclin D, CDK 4 and PCNA was observed with SA treatment corroborating the G1 block. DNA gel electrophoresis showed an oligonucleotide ladder pattern, a distinct characteristic of DNA fragmentation associated with programmed cell death. Ribonuclease protection assay revealed inhibition of bcl2 expression at transcriptional level. SA treatment also resulted in induction of caspase-3 activity. The results suggest the involvement of bcl2 and Caspase-3 in SA induced apoptosis of human U937 cells.

Topics: Apoptosis; Caspase 3; Cell Survival; Cinnamates; DNA Fragmentation; Enzyme Activation; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Naphthoquinones; Neoplasms; Ribonucleases; Tetrazolium Salts; Thiazoles; Transcription, Genetic; U937 Cells

Alkannin and shikonin (A/S) derivatives have been found in the roots of several Boraginaceous species and are produced through plant tissue cultures. The chiral compounds alkannins and shikonins are potent pharmaceutical substances with a wide spectrum of pharmacological activities such as wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant. Although oligomeric A/S derivatives have been detected in root extracts and commercial samples their detection and determination through high-performance liquid chromatography has not been reported. Therefore, in the present study a rapid, simple high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) method was developed to detect, separate and determine monomeric and oligomeric/polymeric derivatives of alkannin/shikonin simultaneously for the first time. An optimization of HPLC-DAD parameters was performed. Both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were applied, in order to compare detection of monomeric and oligomeric A/S. Additionally, oligomeric A/S constituents in several samples were identified and the mode of A/S polymerization was proposed.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Naphthoquinones; Plant Extracts; Plant Roots; Spectrometry, Mass, Electrospray Ionization

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal therapeutic, Zicao, has been shown to exhibit antioxidant and anticancer effects. In this study, its ability to induce apoptosis in cultured Tca-8113 oral cancer cells was studied. Treatment of the Tca-8113 cells with a variety of concentrations of Shikonin (10-40 microm) resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by the loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation and sub-G1 phase accumulation. Furthermore, apoptosis in the Tca-8113 cells was accompanied by the activation of protease caspase-8, -9, -3 and low expression of Bcl-2 protein. Interestingly, inactivation of the NF-kappaB pathway was found in shikonin-induced apoptosis in Tca-8113 cells. These results raise the possibility that the anti-tumor effects of Shikonin in Tca-8113 cells are at least partly through the inactivation of the NF-kappaB pathway and subsequent activation of protease caspase family. Pharmacological inhibition of the NF-kappaB activity by Shikonin might be a powerful treatment option for OSCC in which activation of NF-kappaB plays a critical role in tumor growth and progression.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Squamous Cell; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Gene Expression; Humans; Naphthoquinones; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Time Factors

Shikonin and its derivatives are formed in large amounts in dark-cultured Onosma paniculatum cells. In order to isolate and identify the genes regulating shikonin biosynthesis, we constructed and characterized a full-length-enriched cDNA library of dark-cultured cells by using the SMART (Switching Mechanism At 5'-end of RNA Transcript) cDNA synthesis and LD-PCR (long-distance PCR) strategies. The titer of the primary cDNA library was 1.04 x 10(6)pfu/mL with a recombination rate of 99.60%. Most of the cDNA inserts ranged from 1.0 to 2.5 kb, and 78.33% of the 76 randomly selected clones contained full-length coding regions. Expression analysis of randomly selected genes by small scale microarray revealed that 23 genes were down-regulated, including 17 genes with known functions, 2 genes with putative functions, and 4 novel genes, and that 3 genes were up-regulated (two-fold) in cells cultured under white light as compared with those cultured in the dark. Interestingly, two of the down-regulated genes, encoding aci-reductone dioxygenase (ARD)-like protein and ethylene responsive factor (ERF), are involved in ethylene biosynthesis and signal transduction, implying that ethylene might play an important role as a signal molecule in light-regulated shikonin formation. These data contribute to a better understanding of light-involvement in regulating the formation of plant secondary metabolites.

Topics: Boraginaceae; Cells, Cultured; DNA, Complementary; Electrophoresis, Agar Gel; Ethylenes; Expressed Sequence Tags; Gene Expression Profiling; Gene Expression Regulation, Plant; Gene Library; Genes, Plant; Light; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology, Amino Acid

This work reports the extraction procedures of alkannin/shikonin mixture from roots of six populations of Onosma echioides, by means of three extraction techniques: Soxhlet extraction, maceration and rapid solid-liquid dynamic extraction (RSLDE). Five solvents with different polarity (hexane, petroleum ether, chloroform, ethyl acetate, methanol) were also studied. Analysis of the extracts was performed by an HPLC-DAD (diode array detector) system. The most efficient extraction technique was Soxhlet procedure using ethyl acetate for 6 h. Studied samples of O. echioides showed an alkannin/shikonin content in the range of 0.02-0.24 mg/kg. Other naphthoquinone derivatives (deoxyalkannin/deoxyshikonin and 5,8-dihydroxy-2-(4-methyl-6-oxo-5,6-dihydro-2H-pyran-2-yl)-[1,4]naphthoquinone and arnebin-6) were found for the first time in O. echioides and characterized in the extracts using HPLC-MS apparatus equipped with an ESI ionization source.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Molecular Structure; Naphthoquinones; Plant Extracts; Plant Roots; Solvents; Spectrometry, Mass, Electrospray Ionization

To study the inhibitory effect of shikonin on the proliferation of human airway smooth muscle cells (HASMCs) in vitro.. HASMCs from the trachea were obtained by primary culture of the tissue explants and adherent culture. The HASMCs were exposed to shikonin at 0 (control group), 0.5, 1, 2, 5, 10, 20, and 40 micromol/L for 12, 24, and 48 h, after which the cell proliferation was assessed by 3-(4,5-carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay. Flow cytometry was used for cell cycle analysis of the HASMCs exposed to shikonin at 40, 20, 10, 5 micromol/L and 0 micromol/L (control group) for 24 h. Immunocytochemistry with SP method was performed to detect the expression of proliferating cell nuclear antigen (PCNA) in the HASMCs treated with shikonin at 20 micromol/L and 0 micromol/L (control group) for 24 h.. Shikonin at the concentrations of 20 and 40 micromol/L significantly inhibited the proliferation of the cells (P<0.05), and the effect was especially obvious after 48 h of cell exposure, with inhibition rates of 30.1% and 42.9%, respectively. No significant difference was found between the two concentrations for their cell growth inhibition effects (P>0.05). Shikonin at the concentrations of 20 and 40 micromol/L caused significant cell cycle arrest in G(0)/G(1) phase (P<0.05), the effect of which, however, was not concentration-dependent (P>0.05). Shikohin at 20 micromol/L significantly down-regulated the expression of PCNA in the cells (P<0.05).. Shikonin can inhibit the proliferation of HASMCs in vitro.

Topics: Cell Cycle; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Immunohistochemistry; Muscle, Smooth; Naphthoquinones; Proliferating Cell Nuclear Antigen; Trachea

Three naphthoquinones were isolated by bioassay-guided fractionation from the CHCl(3) extracts of roots of Lithospermum erythrorhizon. They were identified as acetylshikonin (1), isobutyrylshikonin (2), and beta-hydroxyisovalerylshikonin (3) on the basis of their spectroscopic analyses. The compounds 1-3 were tested for their inhibitory activities against human ACAT-1 (hACAT-1) or human ACAT-2 (hACAT-2). Compound 2 preferentially inhibited hACAT-2 (IC(50)=57.5microM) than hACAT-1 (32% at 120microM), whereas compounds 1 and 3 showed weak inhibitory activities in both hACAT-1 and -2. To develop more potent hACAT inhibitor, shikonin derivatives (5-11) were synthesized by semi-synthesis of shikonin (4), which was prepared by hydrolysis of 1-3. Among them, compounds 5 and 7 exhibited the strong inhibitory activities against hACAT-1 and -2. Furthermore, we demonstrated that compound 7 behaved as a potent ACAT inhibitor in not only in vitro assay system but also cell-based assay system.

Topics: Chloroform; Enzyme Inhibitors; Humans; Isoenzymes; Lithospermum; Microscopy, Fluorescence; Naphthoquinones; Plant Roots; Solvents; Sterol O-Acyltransferase; Structure-Activity Relationship; Substrate Specificity

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, mAbs and EGFR tyrosine kinase inhibitors seemed to be the most promising. However they have demonstrated scarce utility in therapy, the former being effective only at toxic doses, the latter resulting inefficient in colon cancer. This paper presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphthoquinone core as shikonin, an agent with great anti-tumor potential. In HT29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 microM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer.

Topics: Apoptosis; Colonic Neoplasms; Dose-Response Relationship, Drug; ErbB Receptors; HT29 Cells; Humans; Microscopy, Confocal; Naphthoquinones; Protein Kinase Inhibitors

We previously developed a gene-gun-based in vivo screening system and identified shikonin as a potent suppressor of tumor necrosis factor-alpha (TNF-alpha) gene expression. Here, we show that shikonin selectively inhibits the expression of TNF-alpha at the RNA splicing level. Treatment of lipopolysaccharide-stimulated human primary monocytes and THP-1 cells with shikonin resulted in normal transcriptional induction of TNF-alpha, but unspliced pre-mRNA accumulated at the expense of functional mRNA. This effect occurred with noncytotoxic doses of shikonin and was highly specific, because mRNA production of neither a housekeeping gene nor another inflammatory cytokine gene, interleukin-8 (IL-8), was affected. Moreover, cotreatment with lipopolysaccharide (LPS) and shikonin increased the endpoint protein production of IL-8, accompanied by suppressed activation of the double-stranded RNA-activated protein kinase (PKR) pathway. Because PKR inactivation has been shown to down-regulate the splicing process of TNF-alpha RNA and interfere with translation, our findings suggest that shikonin may achieve differential modulation of cytokine protein expression through inactivation of the PKR pathway and reveal that regulation of TNF-alpha pre-mRNA splicing may constitute a promising target for future anti-inflammatory application.

Topics: eIF-2 Kinase; Gene Expression; Humans; Interleukin-8; Lipopolysaccharides; Monocytes; Naphthoquinones; RNA Precursors; RNA Splicing; Signal Transduction; Tumor Necrosis Factor-alpha

Shikonin has been reported to induce apoptosis and inhibit angiogenesis in vivo and in vitro. 6-(1-propoxyiminoalkyl)-5,8-dimethoxyoxy 1,4-naphtoquinone S-64 (DMNQ S-64) was synthesized as a shikonin derivative. In this article, the underlying apoptotic mechanism of DMNQ S-64 was examined. DMNQ S-64 exerted cytotoxicity against A549 lung carcinoma cells with IC(50) of 27.3 microM. Apoptotic bodies were observed in DMNQ S-64-treated A549 cells by 4'-6-diamidino-2-phenylindole (DAPI) staining assay. DMNQ S-64 also increased sub-G1 DNA portion in a concentration-dependent manner by flow cytometric analysis. Western blotting has revealed that DMNQ S-64 effectively activates the expression of caspase 8, 9, and 3, cleaves poly (ADP-ribose) polymerase, and increases the ratio of Bax/Bcl-2. Furthermore, cytochrome c was released in a concentration-dependent manner by DMNQ S-64. Similarly, DMNQ S-64 significantly increased caspase 3 activity by enzyme-linked immunosorbent assay (ELISA). It also significantly inhibited the level of prostaglandin E2 (PGE(2)) by ELISA and downregulated the expression of cyclooxygenase-2 (COX-2) in a concentration-dependent manner. Taken together, DMNQ S-64 may exhibit cytotoxicity against A549 cells via caspase activation and COX-2 inhibition.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Enzyme Activation; Humans; Hydroxylamines; Lung Neoplasms; Membrane Proteins; Naphthoquinones

Defect in apoptotic signaling and up-regulation of drug transporters in cancer cells significantly limits the effectiveness of cancer chemotherapy. We propose that an agent inducing non-apoptotic cell death may overcome cancer drug resistance and showed that shikonin, a naturally occurring naphthoquinone, induced a cell death in MCF-7 and HEK293 distinct from apoptosis and characterized with (a) a morphology of necrotic cell death; (b) loss of plasma membrane integrity; (c) loss of mitochondrial membrane potentials; (d) activation of autophagy as a downstream consequence of cell death, but not a contributing factor; (e) elevation of reactive oxygen species with no critical roles contributing to cell death; and (f) that the cell death was prevented by a small molecule, necrostatin-1, that specifically prevents cells from necroptosis. The characteristics fully comply with those of necroptosis, a basic cell-death pathway recently identified by Degterev et al. with potential relevance to human pathology. Furthermore, we proved that shikonin showed a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-x(L), which account for most of the clinical cancer drug resistance. To our best knowledge, this is the first report to document the induction of necroptosis by a small molecular compound to circumvent cancer drug resistance.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Cell Death; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Mice; Mice, Nude; Naphthoquinones; Necrosis; Neoplasm Transplantation

The application of microcapsule for pharmaceutical dosage form for various drugs has received considerable attention in recent years due to its multiple advantages. The most frequently used crosslinking agent formaldehyde in the gelatin-acacia microencapsulation process was altered by glycerol in this study. The effect of various parameters such as the concentration of surfactant, concentration of gelatin and continuous phase pH condition on the microcapsule particle size distribution was experimentally investigated. It was shown that the optimum concentration for surfactant/oil ratio is 1/10 and gelatin/oil ratio is 1/5 in the pH condition of approximately 4-6 for the coacervation process. Results obtained from microscopy observation revealed that one core microcapsule prepared by 6% glycerol was no different from formaldehyde. Hence, glycerol was demonstrated to be a good potential non-toxic crosslinking material for the applications of encapsulated extract containing shikonin.

Topics: Animals; Cattle; Drug Compounding; Drug Delivery Systems; Formaldehyde; Gelatin; Glycerol; Gum Arabic; Naphthoquinones

The root extracts of Onosma leptanhtha were evaluated for their anti-iflammatory and cytotoxic activities. The cyclohexane extract, which appeared as the most active in both assays, has been further subjected to bioassay-directed fractionation to afford the naphthazarine derivatives: beta,beta-dimethylacrylshikonin (1), isovalerylshikonin (2) and acetylshikonin (3). The evaluation of the anti-inflammatory activity was performed on carrageenan-induced rat paw edema test. All the tested compounds proved to be active, while compound 3 showed the best anti-inflammatory effect. In addition, the cytotoxic activity of the extracts and isolated compounds, was also assayed against L1210 murine lymphoblastic leukemia cell line, and human fibrosarcoma HT-1080 cells. Compound 1 exhibited remarkable cytotoxic activity (390 nM for L1210 cells), which is superior to that of shikonin, which was used as control.

Topics: Animals; Anthraquinones; Anti-Inflammatory Agents; Antineoplastic Agents; Biological Assay; Boraginaceae; Carrageenan; Cell Line, Tumor; Cyclohexanes; Edema; Humans; Indomethacin; Inhibitory Concentration 50; Male; Naphthoquinones; Plant Extracts; Plant Roots; Rats; Rats, Wistar

1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enylfuran-2-caroxylate (SH-7), a new naphthoquinone compound, derived from shikonin, exhibited obvious inhibitory actions on topoisomerase II (Topo II) and topoisomerase I (Topo I), which were stronger than its mother compound shikonin. Notably, the SH-7's inhibitory potency on Topo II was much stronger than that on Topo I. In addition, SH-7 significantly stabilized Topo II-DNA cleavable complex and elevated the expression of phosphorylated-H2AX. The in vitro cell-based investigation demonstrated that SH-7 displayed wide cytotoxicity in diversified cancer cell lines with the mean IC(50) value of 7.75 microM. One important finding is SH-7 displayed significant cytotoxicity in the 3 MDR cell lines, with an average IC(50) value nearly equivalent to that of the corresponding parental cell lines. The average resistance factor (RF) of SH-7 was 1.74, which was much lower than those of reference drugs VP-16 (RF 145.92), ADR (RF 105.97) and VCR (RF 197.39). Further studies illustrated that SH-7 had the marked apoptosis-inducing function on leukemia HL-60 cells, which was validated to be of mitochondria-dependence. The in vivo experiments showed that SH-7 had inhibitory effects on S-180 sarcoma implanted to mice, SMMC-7721, BEL-7402 human hepatocellular carcinoma and PC-3 human prostate cancer implanted to nude mice. Taken together, these results suggest that SH-7 induces DSBs as a Topo II inhibitor, which was crucial to activate the apoptotic process, and subsequently accounts for its both in vitro and in vivo antitumor activities. The well-defined Topo II inhibitory activity, antitumor effects particularly with its obvious anti-MDR action, better solubility and less toxicity make SH-7 as a potential antitumor drug candidate for further research and development.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Electrophoresis, Agar Gel; Female; Flow Cytometry; Humans; Leukemia; Liver Neoplasms; Male; Mice; Mice, Nude; Naphthoquinones; Neoplasms; Prostatic Neoplasms; Sarcoma; Topoisomerase II Inhibitors; Transplantation, Heterologous

Arnebia euchroma was grown in a 2-l periodically submerged, airlift bioreactor (PSAB) in which the non-submerged (immobilization culture) and submerged (suspension culture) operations were controlled automatically. PSAB had advantages in improving cell growth, shikonin content, shikonin production and cell aggregation compared with suspension culture. Under the optimal submerged/non-submerged period of 10 min/15 h, the shikonin content (4.6%, w/w) and, cell dry mass (16.8 g/l) were 229 and 26% higher than those in suspension culture.

Topics: Biomass; Bioreactors; Boraginaceae; Cell Division; Culture Techniques; Naphthoquinones; Time Factors

Hairy root cultures of Lithospermum canescens were established using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight lines resulting from infection with A. rhizogenes ATCC 15834 demonstrated sufficient biomass increase and were submitted to further investigations. The contents of acetylshikonin (ACS) and isobutyrylshikonin (IBS) in transformed hairy roots made up ca. 10% of those observed in natural roots of L. canescens (24.35 and 14.48 mg g(-1) DW, respectively). One line, Lc1-D, produced the largest amounts of ACS (2.72 mg g(-1) DW) and IBS (0.307 mg g(-1) DW). Traces of pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots.

Topics: Biomass; Chromatography, High Pressure Liquid; Gibberellins; Lithospermum; Naphthoquinones; Plant Roots; Pyrrolizidine Alkaloids; Spectrophotometry, Ultraviolet

The 1,4-naphthoquinone derivative, shikonin, has been shown to increase glucose uptake by adipocytes and myocytes with minor effects on protein tyrosine phosphorylation in the cells (Biochem Biophys Res Commun 292:642-651, 2002). The present study was performed to examine the mechanism of this action of shikonin. Shikonin inhibited the phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3) phosphatase activity of recombinant phosphatase and tensin homolog deleted on chromosome 10 (PTEN) with an IC50 value of 2.7 microM. Shikonin induced marked accumulation of PtdIns-3,4,5-P3 and activation of protein kinase B (PKB) in Chinese hamster ovary cells expressing insulin receptors. In addition to its effect on PTEN, shikonin was found to inhibit several protein phosphatases in cell-free systems. Its effect on tyrosine phosphorylation in intact cells was far weaker than that of pervanadate, a widely used tyrosine phosphatase inhibitor, despite the observation that the effect of shikonin on PKB was more potent than that of pervanadate. These results suggested that the inhibition of PTEN provides a clue to its potent insulin-like actions. We also found that naphthoquinones, including 1,2-naphthoquinone, inhibit PTEN in the cell-free system, which suggested that the effect on PTEN (and thus the effect on phosphatidylinositol 3-kinase signaling) should be taken into account when examining the pharmacological actions of naphthoquinone derivatives.

Topics: Animals; Cells, Cultured; Cricetinae; Cricetulus; Insulin; Naphthoquinones; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptor, Insulin

Shikonin, isolated from the plant Lithospermum erythrorhizon Sieb. Et Zucc, has been reported to induce apoptosis in several tumor cells. However, such effect of shikonin on human breast cancer cells has not been reported. Thus, in the present study, whether shikonin could induce MCF-7 human breast cancer cell apoptosis was investigated. The results showed that shikonin (2.5-80 microM) induced MCF-7 cell death in a time- and dose-dependent manner, as measured by MTT assay. The IC(50) of a 24 h, 48 h and 72 h time course for MCF-7 cells was 7.4+/-0.4, 6.3+/-0.6 and 3.9+/-0.5 microM, respectively. Cellular morphology observation showed that MCF-7 cells underwent marked apoptotic morphological changes upon treatment with 10 microM shikonin compared with the untreated control. Flow cytometric analysis of shikonin-treated MCF-7 cells showed that the ratio of the apoptotic DNA fragmentation increased in a dose-dependent manner. The present study demonstrated for the first time that the cytotoxic effect of shikonin on MCF-7 cells underwent apoptosis process.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Breast Neoplasms; Cell Proliferation; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Naphthoquinones; Tumor Cells, Cultured

Monomeric alkannin and shikonin (A/S) are potent pharmaceutical substances with a wide spectrum of biological activity and comprise the active ingredients for several pharmaceutical preparations. Therefore, the determination of the impurities, degradation products or byproducts in alkannin and shikonin samples is of great importance. Oligomeric alkannin and shikonin are formed during biosynthesis of these bioactive secondary metabolites in Boraginaceaous root plants, during tissue culture production of A/S, during alkaline hydrolysis of A/S esters and also thermal treatment of A/S. In the present study, a dimeric alkannin/shikonin compound was isolated by size exclusion chromatography from alkannin and shikonin commercial samples and its structure was determined by one- and two-dimensional NMR spectroscopy. The structure of the most abundant oligomeric species in these samples, a dimeric naphthoquinone, was established for the fi rst time, indicating that coupling of the side chain of one naphthoquinone unit with the aromatic ring of a second naphthoquinone leads to dimer formation. This type of coupling allows further oligomerization by leaving one isohexenyl side chain available at the second monomer unit.

Topics: Boraginaceae; Chromatography, Gel; Dimerization; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Polymers

Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.

Topics: Amino Acids, Cyclic; Ethylenes; Glycine; Lithospermum; Naphthoquinones; Plant Shoots; Silver

The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin.. The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4',6'-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.. Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.. Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.

Topics: Apoptosis; Caspases; Cell Proliferation; DNA Damage; Flavonoids; HeLa Cells; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; Phosphorylation; Tumor Suppressor Protein p53; Up-Regulation

Using a new method having been developed for the purpose of quantitative determination for peroxyradicals, the presence of peroxyradicals was proved in cigarette smoke. In brief, peroxyradicals in cigarette smoke were measured by ESR spectrometry coupled to non-reductive scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH). As a result, peroxyradicals were found to be major reactive oxygen species (ROS) since the concentration of peroxyradicals recovered from cigarette smoke was much higher than that of any of other ROS (superoxide and hydroxyl radical) and nitric oxide. Furthermore, several antioxidants (ascorbic acid, reduced glutathione, epigallocatechin gallate, shikonin) were examined for scavenging activity against peroxyradicals in the cigarette smoke. Among them shikonin alone exerted the scavenging activity, suggesting that shikonin is promising antioxidant for cigarette filters because of its effectiveness against broad range of ROS including peroxyradicals, heat resistance, nonvolatility and high affinity to the filter.

Topics: Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Naphthoquinones; Nicotiana; Peroxides; Pigments, Biological; Reactive Oxygen Species; Smoke

Topics: Anti-Inflammatory Agents, Non-Steroidal; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Drugs, Chinese Herbal; Lithospermum; Naphthoquinones

To study the anti-proliferation, pro-apoptosis and cell cycle blocking effects of shikonin on rat vascular smooth muscle cell (VSMC) in vitro.. VSMCs were primarily cultured by explant method from the thoracic aorta of male SD rats. Shikonin of different concentration, 4, 2, 1, 0.5, 0.25, and 0 micromol/L was added. The cell viability was detected by MTT method. Cell growth curve was drawn by trypan blue exclusion method. (3)H-thymidine incorporation was used to calculate the inhibition rate of DNA synthesis. Flow cytometry was used to detect the cell cycle. Cell apoptosis was observed by fluorescence microscopy. Western blotting was performed to detect the expression of different cell apoptosis and cell cycle regulatory proteins, such as cyclin D(1) and E, proliferating cell nuclear antigen (PCNA), p21(waf1/cip1), p27(kip1), and p53.. Compared with control group, shikonin had no obvious cytotoxic effect on cell viability at the concentration of 0.25-1 micromol/L (P > 0.05). While it could inhibit, both time- and dose-dependently, the growth of VSMC, which was predominant of 1 micromol/L at 72 h (1.9 x 10(5)/well vs 5.8 x 10(5)/well, P < 0.05), and DNA synthesis was also significantly inhibited in a time- and dose-dependent manner with inhibition rate varied from 33 to 98% (P < 0.05 or P < 0.01). 1 micromol/L shikonin significantly blocked the cell cycle progression in proliferative VSMC, decreased S, G(2)/M phase (P < 0.05) and increased G(0)/G(1) phase (P < 0.05) to quiescent level with sub-G(1) apoptotic distribution at 48 h (10.9% +/- 0.3%). Shikohin at the concentration of 1-2 micromol/L significantly increased the percentage of apoptotic cells in a time- and dose-dependent manner compared with control group (2.8%-23.7% vs 0.2%-0.4%, P < 0.05), and typical apoptotic nuclear morphological changes were observed. 1 micromol/L shikonin significantly down-regulated cyclin D(1), E and PCNA expression, up-regulated p21(wif1/cip1) expression, and did not obviously influence the p27(kip1) and p53 expression.. Shikonin inhibits the proliferation, promotes the apoptosis and blocks cell cycle progression of VSMC. These effects are associated with the expression changes of cell cycle regulatory proteins.

Topics: Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Drugs, Chinese Herbal; Lithospermum; Male; Muscle, Smooth, Vascular; Naphthoquinones; Rats; Rats, Sprague-Dawley

Natural shikonin compounds and their derivatives have cytotoxicity and antitumor effects. This study was to explore in vitro and in vivo antitumor effects of SYUNZ-7 [2 or 3, 11-bis(phenylsulfanyl)-6-isohexenylnaphthazarin] and the mechanisms.. In vitro antiproliferation effects of SYUNZ-7 on human lung adenocarcinoma cell line GLC-82, human nasopharyngeal cancer cell line CNE2, human oral cavity cancer cell line KB, human gastric cancer cell line MGC-803 and human hepatocellular cancer cell line HepG2 were tested by MTT assay. In vivo antitumor effect of SYUNZ-7 was tested using ascitic cancer EAC xenograft in mice and CNE2 xenograft in nude mice models. Cell apoptosis and cell cycle distribution were assessed by flow cytometry. The in vivo effect of SYUNZ-7 on angiogenesis was detected by immunohistochemistry.. The 50% inhibitory concentrations (IC(50)) of SYUNZ-7 to GLC-82, CNE2, KB, MGC-803, and HepG2 cells were (2.18+/-0.04) microg/ml, (4.17+/-0.09) microg/ml, (5.41+/-0.10) microg/ml, (6.41+/-0.14) microg/ml, and (9.99+/-0.21) microg/ml, respectively. Under the treatment of 1, 2, 4, and 8 mg/kg of SYUNZ-7, the inhibitory rates of EAC xenografts in mice were (40.5+/-0.14)%, (50.9+/-2.3)%, (61.7+/-1.8)%, and (65.6+/-7.4)%, respectively (P<0.01). Under the treatment of 1, 2, and 4 mg/kg of SYUNZ-7, the inhibitory rates of CNE2 xenografts in nude mice were 24.7%, 38.3%, and 41.2%, respectively (P<0.05). SYUNZ-7 induced apoptosis of CNE2 cells in time- and concentration-dependent manners, and blocked the transition of CNE2 cells from S to G(2)/M phase. SYUNZ-7 also inhibited the angiogenesis of CNE2 xenografts in nude mice in a concentration-dependent manner.. SYUNZ-7 has strong in vivo and in vitro antitumor effects which are related to inducing cell apoptosis, blocking cell cycle, and inhibiting angiogenesis of tumor.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Ehrlich Tumor; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Female; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Naphthoquinones; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Neovascularization, Pathologic

Tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNF-alpha promoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNF-alpha promoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNF-alpha promoter activation and also showed significant suppression of transgenic human TNF-alpha mRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNF-alpha promoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNF-alpha by inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNF-alpha promoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics.

Topics: Animals; Anti-Inflammatory Agents; Betamethasone; Blotting, Western; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Gene Transfer Techniques; Humans; Hydrocortisone; Inflammation; Lithospermum; Luciferases; Mice; Mice, Inbred BALB C; Models, Chemical; Naphthoquinones; NF-kappa B; Plasmids; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Time Factors; Transcription Factor RelA; Transcription, Genetic; Transcriptional Activation; Transfection; Transgenes; Tumor Necrosis Factor-alpha

The bioactive compound shikonin was successfully isolated and purified from the crude extract of the traditional Chinese medicinal plant Lithospermum erythrorhizon Sieb. et Zucc. by preparative high-speed counter-current chromatography (HSCCC). The preparative HSCCC was performed using a two-phase solvent system composed of n-hexane-ethylacetate-ethanol-water (16:14:14:5 (v/v)). A total amount of 19.6 mg of shikonin at 98.9% purity was obtained from 52 mg of the crude extract (containing 38.9% shikonin) with 96.9% recovery. The preparative isolation and purification of shikonin by HSCCC was completed in 200 min in a one-step separation.

Topics: Countercurrent Distribution; Lithospermum; Naphthoquinones; Plants, Medicinal

The effects of a naphthoquinone analogue, shikonin/alkannin (SA) and derivatives (acetylshikonin and beta,beta-dimethylacrylshikonin), on vascular reactivity were studied with isolated rat aortic rings. At lower concentrations, SA and its derivatives concentration-dependently inhibit the agonist-induced (acetylcholine and histamine) relaxation in PE precontracted aorta in an endothelium-dependent manner with IC (50) values ranging from 0.2 to 1.5 microM. In addition to the effect on agonist-induced vasorelaxation, the Ca (2+) ionophore A23187-induced vasorelaxation was also inhibited or reversed by SA. However, SA had no effect on sodium nitroprusside-induced (guanylate cyclase activator) vasorelaxation. These data suggested that SA and its derivatives might be acting as inhibitors of nitric oxide synthesis in endothelium. At a concentration greater than 10 microM, SA induced contraction of intact but not denuded aorta which could be inhibited by prior treatment with indomethacin, a cyclooxygenase inhibitor. In summary, the results from this study showed that SA and its derivatives inhibited agonist-induced relaxation at lower concentrations and induced vasocontraction at higher concentrations. All the effects seen with SA were endothelium-dependent, however, through different mechanisms. Abbreviations. SA:shikonin/alkannin PE:phenylephrine Ach:acetylcholine SNP:sodium nitroprusside eNOS:endothelial nitric oxide synthase L-NAME: Nw-nitro- L-arginine methyl ester

Topics: Acetylcholine; Animals; Aorta, Thoracic; Boraginaceae; Dose-Response Relationship, Drug; Endothelium, Vascular; Histamine; Inhibitory Concentration 50; Muscle Contraction; Naphthoquinones; Phytotherapy; Plant Extracts; Plant Roots; Rats; Rats, Wistar

Natural products regulate cell growth in response to oncogene activation that induces cell cycle arrest and apoptosis in tumor cell lines. We investigated the mechanisms of caspase activation in human malignant melanoma, A375-S2 cells, by the natural product shikonin, which was isolated from the plant Lithospermum erythrorhizon SIEB. et ZUCC. Shikonin inhibited cell growth in a time- and dose-dependent manner, which might be mediated through up-regulation of p53 and down-regulation of cyclin-dependent protein kinase 4. Caspase activation was detected in shikonin-induced cell apoptosis, which involved in a post-mitochondrial caspase-9-dependent pathway. Decreased Bcl-2 protein levels and increased Bax protein levels were positively correlated with elevated expression of p53 protein. Apoptosis-inducing factor, another apoptotic protein of mitochondria, partially contributed to shikonin-induced release of cytochrome c. Taken together, shikonin-induced DNA damage activates p53 and caspase-9 pathways.

Topics: Apoptosis; Caspase 9; Caspases; Cell Cycle; Cell Division; Cell Line, Tumor; DNA Damage; Dose-Response Relationship, Drug; G1 Phase; Growth Inhibitors; Humans; Melanoma; Naphthoquinones; Tumor Suppressor Protein p53

Naturally occurring isohexenylnaphthazarins (IHN), such as Alkannin, Shikonin (A/S) and their derivatives, are potent pharmaceutical substances with a wide spectrum of biological activity. In the present study, inclusion complexes of alkannin and shikonin commercial samples and IHN derivatives in the form of an oily extract of Alkanna tinctoria roots were formed with beta-cyclodextrin (CD) and beta-HPCD. These complexes were investigated to evaluate the effect of complexation on their aqueous solubility, decoloration, and also the percentage of polymeric A/S and IHN derivatives enclosed in the CDs cavity, since these decrease the active monomeric IHN. Both beta-CD and beta-HPCD increased the aqueous solubility of A/S and IHN derivatives and thus inclusion complexes can be used as drug delivery systems for A/S in both internal (capsules, tablets) and external hydrophilic pharmaceutical and cosmetic preparations (creams, gels, sprays) with enhanced bioavailability. The inclusion complexes formed had a pale purple colour, contributing to the partial decoloration of the A/S and thus of the fi nal pharmaceutical preparations. Finally, CDs selectively included more monomeric and less polymeric IHN, compared with the initial each time sample that is encapsulated; thus inclusion complexes may present enhanced biological activity.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; beta-Cyclodextrins; Biological Availability; Capsules; Chemistry, Pharmaceutical; Cyclodextrins; Dosage Forms; Drug Carriers; Microscopy, Electron; Naphthoquinones; Plant Extracts; Solubility

Shikonin, isolated from the plant Lithospermum erythrorhizon Sieb. ET Zucc, inhibited tumor cell growth and induced cell death in various tumor cells, with 50% growth inhibition of human cervical cancer cells, HeLa, at 18.9 +/- 1.1 mumol L-1. Treated with 40 mumol L-1 shikonin, HeLa cells underwent marked apoptotic morphological changes such as a round shape, membrane blebbing and apoptotic bodies derived from the fragmented nuclei. Another hallmark of apoptosis, DNA fragmentation, was observed by gel electrophoresis. Shikonin (10 mumol L-1) significantly blocked the transition from G1 to S phase in the HeLa cell cycle. Pan-caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK) or caspase-8 inhibitor (Z-IETD-FMK) effectively inhibited shikonin-induced cell death, while caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-9 inhibitor (Z-LEHD-FMK) failed to affect cell death. Caspase-3 activity significantly increased within 12 h after shikonin treatment. Reduced expression of inhibitor of caspase-activated deoxyribonuclease (ICAD) after exposure to shikonin for 12 h suggests the resultant activation of caspase-activated deoxyribonuclease (CAD), leading to apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 3; Caspase Inhibitors; Cell Division; Cell Line, Tumor; DNA Fragmentation; Flow Cytometry; HeLa Cells; Humans; Inhibitory Concentration 50; Lithospermum; Mice; Naphthoquinones; Phytotherapy

To explore cultural conditions of shikonin production by cell cultures of Lithospermum erythrorhizon.. Orthogonal design was applied in determination of shikonin within the medium. Flask test was applied in the study of shikonin production by the amount of ventilation.. The best medium consisted of 100 mg/L L-phenylalanine, 2 mg/L IAA and 800 mg/L Ca(NO3)2 4H2O. The best amount of ventilation was get by shaken at 150 r/min.. This test provided data for producing shikonin by cell cultures of Lithospermum erythrorhizon.

Topics: Cell Culture Techniques; Cells, Cultured; Culture Media; Indoleacetic Acids; Lithospermum; Naphthoquinones; Phenylalanine; Plants, Medicinal; Time Factors; Ventilation

The immunomodulatory activity of acetylshikonin (ACS) and isobutyrylshikonin (IBS) was studied in female and male inbred Balb/c mice, and in F1 hybrids (Balb/c x C3H). ACS and IBS were isolated from Lithospermum canescens Lehm. (Boraginaceae) roots. Splenocytes from mice fed 40 microg of ACS had higher proliferative potential in cultures with PHA than corresponding controls and also higher migratory in vitro activity than splenocytes obtained from control animals. ACS at a 40 microg daily dose stimulated G-v-H reaction but inhibited it at a 200 microg dose. IBS at a 40 microg dose significantly increased humoral response.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Cell Division; Erythrocytes; Female; Graft vs Host Reaction; Immunity, Cellular; Lithospermum; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Naphthoquinones; Plant Roots; Sheep; Spleen

The chiral pair alkannin and shikonin (A/S) and their isohexenylnaphthazarin (IHN) esters, which are naturally occurring hydroxynaphthoquinones (HNQ), are potent pharmaceutical substances with a wide spectrum of biological activity. The stability of A/S and their derivatives during process and storage is crucial to their use as drugs, cosmetics and food additives. The influence of alkaline media and of IHN esters hydrolysis was experimentally investigated on IHN polymerization by size exclusion chromatography (SEC). It was proved that during IHN esters hydrolysis, polymeric A/S and IHN are formed. An optimization of the hydrolysis conditions of IHN esters was also approached in terms of polymerization. Hydrolysis of IHN from a pure mixture of pigments proved preferable to that of preliminary root extracts by means of IHN polymerization, even for analytical determination; non-polar solvents are proposed for the extraction of IHN from roots, followed by hydrolysis, aiming to minimize the polymeric IHN and A/S formed. It was also proved that polymerization of IHN in alkaline media and during hydrolysis of IHN esters proceeds through the intermediate formation of semiquinones; after acidification, coupling of semiquinones with phenoxyl radicals results in polymeric IHN structures.

Topics: Boraginaceae; Chromatography, Gel; Cosmetics; Drug Stability; Esters; Food Additives; Hydrogen-Ion Concentration; Hydrolysis; Naphthoquinones; Pharmaceutical Preparations; Plant Roots; Polymers

Polymerization of naturally occurring isohexenylnaphthazarins (IHN), such as alkannin, shikonin (A/S) and their derivatives, which are potent pharmaceutical substances, significantly affects their use in pharmaceuticals, cosmetics and as food colorants, because it leads to reduction of the lustre of their red coloration, a decrease in their solubility and reduces the active monomeric IHN derivatives. In the present study, the influence of several crucial variables (processing and storage) was experimentally investigated on IHN polymerization by size exclusion chromatography (SEC). Temperature and solvent polarity increased significantly the concentration of hydroxynaphthoquinone (HNQ) polymers, while air and light exposure conditions did not significantly affect IHN polymerization. Low temperatures are proposed for all processes of industrial production of pharmaceutical preparations containing IHN and HNQ. An optimization of the industrial conditions used for the preparation of pharmaceutical and cosmetic preparations containing IHN, maximizing the active monomeric IHN fraction, was performed.

Topics: Air; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Light; Models, Molecular; Molecular Structure; Naphthoquinones; Polymers; Solvents; Temperature

The chiral pair alkannin and shikonin (A/S) are potent pharmaceutical substances with a wide spectrum of biological activity; their enantiomeric ratio does not influence the major biological activity studied hitherto. Nevertheless, in pharmaceutical development and approval of chiral drugs from the Health and Regulatory Authorities, full documentation of methods of analysis of enantiomeric drugs, is required in order to evaluate the enantiomeric purity of starting materials and final products and to control the stability of enantiomers in pharmaceutical formulations under several experimental conditions. In the present study, the enantiomeric ratio of A/S was determined in several commercial samples of alkannin and shikonin and also the proportion of A/S derivatives in several Alkanna root samples, which are all used as active ingredients in pharmaceuticals. Light and air proved not to influence the enantiomeric ratio of A/S on a shikonin commercial sample, and temperature also did not alter the A/S ratio on shikonin and alkannin commercial samples. Microencapsulation of alkannin and shikonin commercial samples in ethylcellulose microspheres and also molecular inclusion of a shikonin commercial sample in beta-hydroxypropyl-cyclodextrin, which are used as drug delivery systems, did not alter the A/S enantiomeric ratio.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Capsules; Chromatography, High Pressure Liquid; Naphthoquinones; Pharmaceutical Preparations; Stereoisomerism; Temperature

Shikonin is a main constituent of the roots of Lithospermum erythrorhizon that has antimutagenic activity. However, its other biological activities are not well-known. Shikonin displayed a strong inhibitory effect against human colorectal carcinoma COLO 205 cells and human leukemia HL-60 cells, with estimated IC(50) values of 3.12 and 5.5 microM, respectively, but were less effective against human colorectal carcinoma HT-29 cells, with an estimated IC(50) value of 14.8 microM. Induce apoptosis was confirmed in COLO 205 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by loss of mitochondrial membrane potential, reactive oxygen species (ROS) generation, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor (DFF-45) were accompanied by activation of caspase-9 and -3 triggered by shikonin in COLO 205 cells. Here, we found that shikonin-induced apoptotic cell death was accompanied by upregulation of p27, p53, and Bad and down-regulation of Bcl-2 and Bcl-X(L), while shikonin had little effect on the levels of Bax protein. Taken together, we suggested that shikonin-induced apoptosis is triggered by the release of cytochrome c into cytosol, procaspase-9 processing, activation of caspase-3, degradation of PARP, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by shikonin may provide a pivotal mechanism for its cancer chemopreventive action.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 9; Caspases; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; DNA Fragmentation; Enzyme Activation; Humans; Membrane Potentials; Mitochondria; Naphthoquinones; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Culture Techniques; DNA, Complementary; Enzymes; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Lithospermum; Molecular Sequence Data; Naphthoquinones; Plant Proteins; Propanols; RNA, Messenger; Sequence Analysis, DNA; Substrate Specificity

Naturally occurring gums and resins with beneficial pharmaceutical and nutraceutical properties were tested for their possible protective effect against copper-induced LDL oxidation in vitro. Chiosmastic gum (CMG) (Pistacia lentiscus var. Chia resin) was the most effective in protecting human LDL from oxidation. The minimum and maximum doses for the saturation phenomena of inhibition of LDL oxidation were 2.5 mg and 50 mg CMG (75.3% and 99.9%, respectively). The methanol/water extract of CMG was the most effective compared with other solvent combinations. CMG when fractionated in order to determine a structure-activity relationship showed that the total mastic essential oil, collofonium-like residue and acidic fractions of CMG exhibited a high protective activity ranging from 65.0% to 77.8%. The other natural gums and resins (CMG resin 'liquid collection', P. terebinthus var. Chia resin, dammar resin, acacia gum, tragacanth gum, storax gum) also tested as above, showed 27.0%-78.8% of the maximum LDL protection. The other naturally occurring substances, i.e. triterpenes (amyrin, oleanolic acid, ursolic acid, lupeol, 18-a-glycyrrhetinic acid) and hydroxynaphthoquinones (naphthazarin, shikonin and alkannin) showed 53.5%-78.8% and 27.0%-64.1% LDL protective activity, respectively. The combination effects (68.7%-76.2% LDL protection) of ursolic-, oleanolic- and ursodeoxycholic- acids were almost equal to the effect (75.3%) of the CMG extract in comparable doses.

Topics: Cholesterol, LDL; Dose-Response Relationship, Drug; Gum Arabic; Humans; Karaya Gum; Mastic Resin; Naphthoquinones; Oils, Volatile; Oxidation-Reduction; Pigments, Biological; Pistacia; Plant Extracts; Resins, Plant; Structure-Activity Relationship; Triterpenes

Shikonin is a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine with various biological activities, including inhibition of human immunodeficiency virus (HIV) type 1 (HIV-1). G protein-coupled chemokine receptors are used by HIV-1 as coreceptors to enter the host cells. In this study, we assessed the effects of shikonin on chemokine receptor function and HIV-1 replication. The results showed that, at nanomolar concentrations, shikonin inhibited monocyte chemotaxis and calcium flux in response to a variety of CC chemokines (CCL2 [monocyte chemoattractant protein 1], CCL3 [macrophage inflammatory protein 1alpha], and CCL5 [regulated upon activation, normal T-cell expressed and secreted protein]), the CXC chemokine (CXCL12 [stromal cell-derived factor 1alpha]), and classic chemoattractants (formylmethionyl-leucine-phenylalanine and complement fraction C5a). Shikonin down-regulated surface expression of CCR5, a primary HIV-1 coreceptor, on macrophages to a greater degree than the other receptors (CCR1, CCR2, CXCR4, and the formyl peptide receptor) did. CCR5 mRNA expression was also down-regulated by the compound. Additionally, shikonin inhibited the replication of a multidrug-resistant strain and pediatric clinical isolates of HIV in human peripheral blood mononuclear cells, with 50% inhibitory concentrations (IC(50)s) ranging from 96 to 366 nM. Shikonin also effectively inhibited the replication of the HIV Ba-L isolate in monocytes/macrophages, with an IC(50) of 470 nM. Our results suggest that the anti-HIV and anti-inflammatory activities of shikonin may be related to its interference with chemokine receptor expression and function. Therefore, shikonin, as a naturally occurring, low-molecular-weight pan-chemokine receptor inhibitor, constitutes a basis for the development of novel anti-HIV therapeutic agents.

Topics: Anti-HIV Agents; Anti-Inflammatory Agents; Calcium Signaling; Cell Survival; Cells, Cultured; Chemotaxis, Leukocyte; Down-Regulation; Drugs, Chinese Herbal; Flow Cytometry; Humans; Naphthoquinones; Receptors, CCR5; Receptors, Chemokine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Signal Transduction; Virus Replication

Shikonin isolated from the roots of the Chinese herb Lithospermum erythrorhizon has been associated with anti-inflammatory properties. We evaluated shikonin's chemotherapeutic potential and investigated its possible mechanism of action in a human cutaneous neoplasm in tissue culture. Shikonin preferentially inhibits the growth of human epidermoid carcinoma cells concentration- and time-dependently compared to SV-40 transfected keratinocytes, demonstrating its anti-proliferative effects against this cancer cell line. Additionally, shikonin decreased phosphorylated levels of EGFR, ERK1/2 and protein tyrosine kinases, while increasing phosphorylated JNK1/2 levels. Overall, shikonin treatment was associated with increased intracellular levels of phosphorylated apoptosis-related proteins, and decreased levels of proteins associated with proliferation in human epidermoid carcinoma cells.

Topics: Carcinoma, Squamous Cell; Cell Division; Enzyme Inhibitors; ErbB Receptors; Humans; MAP Kinase Signaling System; Naphthoquinones; Phosphorylation; Protein-Tyrosine Kinases; Signal Transduction; Tumor Cells, Cultured

Shikonin has been demonstrated to exhibit anti-cancer activity, but the underlying mechanisms are poorly understood. In this report, we showed that the administration of shikonin could result in the induction of apoptotic cell death of human hepatoma cell line, SK-Hep-1. As evident by the flow-cytometric studies, shikonin has the capability of generating increased amounts of intracellular reactive oxygen species (ROS) during the early stage of this apoptotic process (ca. one-hour), and subsequently accompanied by the dissipation of mitochondrial transmembrane potential (deltapsi (m)) at 3 hours. Further studies indicated that this apoptotic process could effectively be protected by the pretreatment of shikonin-treated cells with glutathione (GSH) and N-acetylcysteine (NAC), a precursor of GSH, but not by cyclosporin A (CyA), an inhibitor of mitochondrial permeability transition (MPT) pore. These data further proved that ROS-mediated oxidative stress was the pivotal element involved in the induction of apoptosis of SK-Hep-1 cells. Taken together, we suggest that shikonin-induced apoptosis of SK-Hep-1 cells proceeds by an oxidative stress-mediated pathway.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; DNA Fragmentation; Flow Cytometry; Humans; In Situ Nick-End Labeling; Lithospermum; Mitochondria; Naphthoquinones; Phytotherapy; Plant Roots; Reactive Oxygen Species

Shikonin (beta-alkannin), a naphthoquinone compound, was found to induce apoptotic features such as chromatin condensation, DNA fragmentation, and activation of caspase 3 in HL60 cells. The mechanism was examined in terms of oxidative stress in the cells. Exposure of the cells to shikonin greatly reduced the total thiols, protein thiols, and glutathione levels, however, lipid peroxide levels were enhanced. The depletion of thiol levels in the cells was thus thought to induce lipid peroxidation and DNA fragmentation. An electron spin resonance study revealed that shikonin reacts directly with glutathione and other oxidative stress-relevant compounds in the lysate of HL60 cells. Pretreatment of such cells with N-acetylcysteine before shikonin treatment completely inhibited the DNA fragmentation. From these results, it was proposed that the chemical reaction between shikonin and cellular thiols such as glutathione and protein thiols induces apoptosis in HL60 cells.

Topics: Acetylcysteine; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspases; DNA Fragmentation; Enzyme Activation; HL-60 Cells; Humans; Lipid Peroxides; Naphthoquinones; Sulfhydryl Compounds

Cell suspension cultures of Lithospermum erythrorhizon produced a large amount of lithospermic acid B, a caffeic acid tetramer, as well as shikonin derivatives (each ca. 10% of dry wt.) when cultured in shikonin production medium M-9. Various culture factors for increasing the production of lithospermic acid B were investigated. Lithospermic acid B production was inhibited by 2, 4-D or NH4+, whereas it was stimulated by Cu2+. These regulatory patterns were similar to those for the production of shikonin derivatives in these cell cultures, suggestive of close relations and similar metabolic regulation between the production of these compounds. Cultivation under light illumination, however, showed that these metabolisms were independently regulated. In particular, blue light showed a stimulatory effect on lithospermic acid B production, while shikonin production was strongly inhibited, indicative of an effective condition for lithospermic acid B production.

Topics: Benzofurans; Cell Culture Techniques; Depsides; Lithospermum; Naphthoquinones; Plant Structures

Shikonin, a red naphthoquinone pigment, is produced by cell cultures of Lithospermum erythrorhizon (Boraginaceae). It is biosynthetically derived from two key precursors, 4-hydroxybenzoate (4HB) and geranyldiphosphate (GPP). The bacterial ubiC gene, encoding chorismate pyruvate-lyase (CPL) which converts chorismate to 4-hydroxybenzoate, was expressed in L. erythrorhizon under the control of the strong (ocs)(3)mas-promoter. This introduced an efficient biosynthetic pathway to 4HB, i.e. a one-step reaction from chorismate, in addition to the endogeneous multi-step phenylpropanoid pathway. Feeding experiments with [1,7-(13)C(2)]shikimic acid showed that in the most active transgenic line, 73% of 4HB was synthesized via the genetically introduced pathway. However, there was no correlation between CPL activity and 4HB glucoside or shikonin accumulation in the transgenic lines. HMG-CoA reductase (HMGR) is involved in the biosynthesis of GPP in L. erythrorhizon. Two forms of HMGR1 of Arabidopsis thaliana were expressed in Lithospermum under control of the (ocs)(3)mas promoter. Only moderate increases in enzyme activity were obtained with the complete enzyme, but high activity was achieved using the soluble cytosolic domain of HMGR1. Shikonin accumulation remained unchanged even upon high expression of soluble HMGR.

Topics: Arabidopsis; Blotting, Northern; Blotting, Southern; Carbon Isotopes; Cell Surface Extensions; Cloning, Molecular; Culture Techniques; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genetic Vectors; Glucosides; Hydroxymethylglutaryl CoA Reductases; Lithospermum; Magnetic Resonance Spectroscopy; Naphthoquinones; Oxo-Acid-Lyases; Parabens; Plant Roots; Plants, Genetically Modified; Polyisoprenyl Phosphates; Shikimic Acid

Alkannin and shikonin, two natural products from Alkanna tinctoria and Lithospermum erhythrorhizon (Boraginaceae), are used in folk medicine where they are claimed to possess, among other properties, wound healing and anti-inflammatory activity. We investigated, together with the structurally related naphthazarin, their in vitro antioxidant and hydroxyl radical scavenging activity as well as their in vivo antiinflammatory activity. I was found that all examined compounds significantly inhibited in vitro lipid peroxidation of ra hepatic microsomal membranes, competed with DMSO for free hydroxyl radicals, and reduced inflammation (mouse paw edema induced by FCA) very efficiently. The examined compounds proved equal or superior to the common reference compounds for each of these properties. I is concluded that the claimed and/or proven actions of alkannin and shikonin are attributable at least partly to their intervention in free radical processes.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Dimethyl Sulfoxide; Edema; Female; Free Radical Scavengers; Freund's Adjuvant; Lipid Peroxidation; Naphthoquinones; Rats; Rats, Inbred F344

Bioassay-directed fractionation of extract of Arnebia euchroma led to the isolation of alkannin (1), shikonin (2), and their derivatives (3-8) as the active principles against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The stereochemistry of alpha-methylbutyryl alkannin (8) is revealed for the first time, and the antimicrobial activity of 8 was compared with its corresponding diastereomer (9). The derivatives 3-9 showed stronger anti-MRSA activity [minimum inhibitory concentrations (MICs) ranged from 1.56 to 3.13 microg/mL] than alkannin or shikonin (MIC = 6.25 microg/mL). Anti-MRSA activity of derivatives was bactericidal with minimum bactericidal concentration (MBC)/MIC < or = 2. In a time-kill assay, the bactericidal activity against MRSA was achieved as rapidly as 2 h. The derivatives 3-9 were also active against vancomycin-resistant Enterococcus faecium (F935) and vancomycin-resistant Enterococcus faecalis (CKU-17) with MICs similar to those with MRSA. Aromatic ester derivatives were also synthesized for antimicrobial activity comparison. None of these compounds were active against Gram-negative bacteria tested. Their cytotoxicity was also evaluated on selected cancer cell lines, and they expressed their activity in the range 0.6-5.4 microg/mL (CD(50)). Our results indicate that the ester derivatives of alkannin are potential candidates of anti-MRSA and anti-VRE agents with antitumor activity.

Topics: Anti-Bacterial Agents; Boraginaceae; Carcinoma, Hepatocellular; China; Drug Screening Assays, Antitumor; Electron Spin Resonance Spectroscopy; Enterococcus faecalis; Female; HeLa Cells; Humans; Methicillin Resistance; Microbial Sensitivity Tests; Molecular Structure; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Ovarian Neoplasms; Plant Bark; Plants, Medicinal; Staphylococcus aureus; Stereoisomerism; Tumor Cells, Cultured; Vancomycin

Beta-hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from Lithospermium radix, most efficiently induced cell-death in two lines of lung cancer cells, namely, NCI-H522 and DMS114, whereas shikonin was effective against a wide variety of tumor cell lines. During our studies of the mechanism of action of beta-HIVS on tumor cells, we found that this compound inhibited protein tyrosine kinase (PTK) activity. The tyrosine kinase activities of a receptor for EGF (EGFR) and v-Src were strongly inhibited and that of KDR/Flk-1 was weakly inhibited by beta-HIVS. The inhibition by beta-HIVS of the activities of EGFR and v-Src was much stronger than that by shikonin. The IC50 values of beta-HIVS for EGFR and v-Src were approximately 0.7 microM and 1 microM, respectively. Moreover, the inhibition of v-Src by beta-HIVS was non-competitive with respect to ATP. These results strongly suggest that the action of beta-HIVS, as well as that of shikonin, involves the inhibition of PTK, and they also suggest the possibility of producing a novel group of PTK inhibitors based on shikonin as the parent compound.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Cell Death; Dose-Response Relationship, Drug; Enzyme Inhibitors; ErbB Receptors; Humans; Inhibitory Concentration 50; Kinetics; Mice; Models, Chemical; Naphthoquinones; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Two cDNAs encoding geranyl diphosphate:4-hy- droxybenzoate 3-geranyltransferase were isolated from Lithospermum erythrorhizon by nested PCR using the conserved amino acid sequences among polyprenyl- transferases for ubiquinone biosynthesis. They were functionally expressed in yeast COQ2 disruptant and showed a strict substrate specificity for geranyl diphosphate as the prenyl donor, in contrast to ubiquinone biosynthetic enzymes, suggesting that they are involved in the biosynthesis of shikonin, a naphthoquinone secondary metabolite. Regulation of their expression by various culture conditions coincided with that of geranyltransferase activity and the secondary metabolites biosynthesized via this enzyme. This is the first established plant prenyltransferase that transfers the prenyl chain to an aromatic substrate.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cloning, Molecular; DNA Primers; Kinetics; Lithospermum; Molecular Sequence Data; Naphthoquinones; Phenotype; Phylogeny; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Homology, Amino Acid

This work demonstrates the use of low-energy ultrasound (US) to enhance secondary metabolite production in plant cell cultures. Suspension culture of Lithospermum erythrorhizon cells was exposed to low-power US (power density < or = 113.9 mW/cm(3)) for short periods (1-8 min). The US exposure significantly stimulated the shikonin biosynthesis of the cells, and at certain US doses, increased the volumetric shikonin yield by about 60%-70%. Meanwhile, the shikonin excreted from the cells was increased from 20% to 65%-70%, due partially to an increase in the cell membrane permeability by sonication. With combined use of US treatment and in situ product extraction by an organic solvent, or the two-phase culture, the volumetric shikonin yield was increased more than two- to threefold. Increasing in the number of US exposures during the culture process usually resulted in negative effects on shikonin yield but slight stimulation of shikonin excretion. US at relatively high energy levels caused slight cell growth depression (maximum 9% decrease in dry cell weight). Two key enzymes for the secondary metabolite biosynthesis of cells, phenylalanine ammonia lyase and p-hydroxybenzoic acid geranyltransferase, were found to be stimulated by the US. The US stimulation of secondary metabolite biosynthesis was attributed to the metabolic activity of cells activated by US, and more specifically, the defense responses of plant cells to the mechanical stress of US irradiation.

Topics: Cells, Cultured; Lithospermum; Naphthoquinones; Permeability; Reproducibility of Results; Sensitivity and Specificity; Time Factors; Ultrasonics

Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor. However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation.

Topics: 3T3 Cells; Adipocytes; Androstadienes; Animals; Anti-Inflammatory Agents, Non-Steroidal; Biological Transport; Drugs, Chinese Herbal; Enzyme Inhibitors; Genistein; Glucose; Glucose Transporter Type 4; Humans; Insulin; Male; Medicine, Chinese Traditional; Mice; Molecular Structure; Monosaccharide Transport Proteins; Muscle Proteins; Myocardium; Naphthoquinones; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptor, Insulin; Signal Transduction; Vanadates; Wortmannin

In an effort to develop new drugs preventing the growth of human immunodeficiency virus (HIV), we developed an in vitro assay method of ribonuclease H (RNase H) activity associated with reverse transcriptase (RT) from HIV-1. Some naphthoquinones, such as 1,4-naphthoquinone (1), vitamin K(3) (2), juglone (3) and plumbagin (6), moderately inhibited RNase H activity, and others, including naphthazarin (5) and shikonins (8-9, 18-23), showed weak inhibition. Diterpenoid quinones, tanshinones (24-28), had also moderate inhibition against RNase H activity. Of these quinones, compound 1 showed the most potent inhibition on RNase H activity with a 50% inhibitory concentration (IC(50)) of 9.5 microM, together with moderate inhibition against RNA-dependent and DNA-dependent DNA polymerase (RDDP and DDDP) activities with IC(50) values of 69 and 36 microM, respectively. Compounds 3 and 5 showed significant inhibition against RDDP (IC(50) = 8 and 10 microM, respectively) and DDDP (IC(50) = 5 and 7 microM, respectively) activities. The structure-activity relationship of the naphthoquinones suggested that non-hydroxylated naphthoquinones (1 and 2) showed significant inhibition of RNase H activity, whereas 5-hydroxylated naphthoquinones (3 and 5) showed potent inhibition against RDDP and DDDP activities.

Topics: Dose-Response Relationship, Drug; HIV Infections; HIV Protease Inhibitors; HIV Reverse Transcriptase; HIV-1; Humans; Inhibitory Concentration 50; Naphthoquinones; Phytotherapy; Quinones; Ribonuclease H

We synthesized DL-shikonin, shikonin, alkanin, and their cyclo-derivatives and acyl-derivatives. These compounds have low cytotoxicity, as well as inhibitory activity against the telomerase enzyme, except cyclo-derivatives.

Topics: Drug Design; Enzyme Inhibitors; Humans; Kinetics; Models, Molecular; Molecular Conformation; Naphthoquinones; Spectrophotometry; Structure-Activity Relationship; Telomerase

Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities.

Topics: Amino Acid Sequence; Asteraceae; Base Sequence; Darkness; Gene Expression Regulation, Plant; Molecular Sequence Data; Naphthoquinones; Plant Proteins; Plant Roots; Restriction Mapping; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Transcription, Genetic; Transformation, Genetic

Shikonin is a chemically characterized component of traditional Chinese herbal medicine and has been shown to possess antiinflammatory activities. We ascertained that shikonin blocked radiolabelled Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) and macrophage inflammatory protein-1 (MIP-1alpha) binding to human monocytes with IC50 values of 3.58 x 10(-6) and 2.57 x 10(-6) M, respectively. In contrast, up to 1.7 x 10(-5) M of shikonin failed to inhibit stromal cell-derived factor-1 (SDF-1alpha) binding to the cells. Additionally, shikonin blocked RANTES and MIP-1alpha binding to stable CC chemokine receptor-1 (CCR1) transfected human embryonic kidney (HEK)/293 cells with IC50 values of 2.63 x 10(-6) and 2.57 x 10(-6) M, respectively. However, shikonin inhibited neither RANTES nor MIP-1alpha binding to CCR5 transfected HEK/293 cells. Shikonin also did not inhibit monocyte chemoattractant protein-1 (MCP-1) binding to CCR2 cells, eotaxin binding to CCR3 cells, interferon-inducible T cell alpha-chemoattractant (I-TAC) binding to CXCR3 cells and SDF-1alpha binding to CXCR4 cells. Additionally, shikonin inhibited RANTES-induced CCR1 cell migration, but did not inhibit CCR1 cell migration induced by epidermal growth factor (EGF). Our study suggests shikonin may be a target for the future design of more potent, highly selective therapeutics that could be useful antiinflammatory agents for selectively blocking the binding of CCR1 ligands.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Chemokine CCL2; Chemokine CCL5; Chemokines; Chemokines, CC; Humans; Naphthoquinones; Receptors, CCR1; Receptors, Chemokine

Studies were conducted with a BK-39 callus culture of Lithospermum erythrorhizon, which produced seven shikonin derivatives (acetylshikonin, propionylshikonin, isobutyrylshikonin, beta,beta-dimethylacrylshikonin, isovalerylshikonin, beta-hydroxyisovalerylshikonin and alpha-methyl-n-butyrylshikonin). A selection of cell aggregates of BK-39 culture on a medium containing p-fluorophenylalanine (PFP) yields a cell line possessing a higher resistance to the inhibitor than the initial culture. Selected BK-39F cultures produced almost the same profile of shikonin naphthoquinones as the initial culture. The shikonin derivative content of PFP-resistant culture was approximately two times higher than that of the control, reaching 12.6% of DW cell biomass.

Topics: Cells, Cultured; Humans; Magnoliopsida; Naphthoquinones; p-Fluorophenylalanine; Plant Roots; Plants, Medicinal

"The extract of shikon" (SK) and shikonin play important roles in the development of granulomatous tissue formation. To reveal the augmenting effect of SK or shikonin on vascular endothelial growth factor (VEGF) production and neovascularization, we investigated murine granulomatous tissue induced by SK and shikonin, comparing them to pouches in which trehalose 6,6'-dimycolate (TDM) was injected. The development of granulomatous tissue formation was evaluated by the wet weight of pouch walls. At day 5 and 7 after SK and shikonin injection, prominent granulomatous tissue formation was detected. Histological observations on the development of granulomatous tissue showed that the pouch was formed in the submuscular connective tissue and necrotic tissue directly facing the cavity and granulomatous tissue developed in the connective tissue. At day 1, VEGF-positive neutrophils accumulated in the pouch wall. Granulomatous tissue formation and neovascularization by injection of SK or shikonin was not more prominent than TDM. However, the present results indicate that SK and shikonin induce neovascularization in granulomatous tissue.

Topics: Animals; CD3 Complex; Endothelial Growth Factors; Flow Cytometry; Granuloma; Lymphokines; Macrophage-1 Antigen; Male; Mice; Mice, Inbred ICR; Naphthoquinones; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Cyclooxygenase-2 (COX-2) is a recognized target for cancer prevention and possibly treatment. To identify novel inhibitors of COX-2, we developed a high throughput reporter gene assay that utilizes a region of the human COX-2 promoter to drive luciferase expression. A total of 968 extracts from 266 plants were screened. Extracts from 12 plants (4.5%), including Arnebia euchroma, a medicinal plant used in the Far East to treat inflammation, inhibited the stimulation of COX-2 promoter activity. The gene promoter assay then was used to identify shikonin, a compound with known anti-inflammatory and chemopreventive properties, as an active compound in A. euchroma. To complement the gene promoter studies, we determined the effects of a mixture of shikonins on phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 in transformed human mammary epithelial cells. Shikonins inhibited PMA-mediated induction of COX-2 mRNA, protein, and prostaglandin E(2) synthesis. In transient transfections, PMA caused a severalfold increase in COX-2 promoter activity, an effect that was suppressed by shikonins. Shikonins also inhibited PMA-mediated stimulation of extracellular signal-regulated kinase1/2 mitogen-activated protein kinases and activator protein-1 activity. Collectively, these results demonstrate the successful development and use of a high throughput reporter gene assay for the identification of a novel inhibitor of COX-2 expression.

Topics: Antineoplastic Agents, Phytogenic; Blotting, Northern; Blotting, Western; Breast; Cell Line, Transformed; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Inhibitors; Epithelial Cells; Humans; Isoenzymes; Luciferases; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Naphthoquinones; Plant Extracts; Plasmids; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Effects of space flight on growth and biosynthetic features of plant cells were studied in two strains of ginseng (Panax ginseng) differing in growth and particularly biosynthetic activities, a strain of Lithospermum Erythrorhizon and a strain of Macrotomia Euchroma which produce biologically active naphroquin-derived pigments. --and also differ in growth and biosynthetic properties. Following exposure aboard MIR and a Space shuttle, cells of the callosal cultures were subjected to callosal or suspension passaging. Biomass yield and biologically active substances--ginseng saponins ginsenoids and shikonin were determined in the cells cultures. There was no evidence for the biomass yield to be significantly altered by space flight; however, the content of biologically active substances was materially changed with the strain.

Topics: Extraterrestrial Environment; Naphthoquinones; Panax; Pigments, Biological; Plants, Medicinal; Seeds; Space Flight

Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin. The cells also produced a related dimer, (+)-rabdosiin. In Linsmaier-Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed. Lithospermic acid, a caffeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures. In the aerial parts of L. erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid.

Topics: Benzofurans; Caffeic Acids; Cells, Cultured; Depsides; Dimerization; Lignans; Magnetic Resonance Spectroscopy; Naphthoquinones; Plants

A high-performance liquid chromatography (HPLC) analysis system based on a water-acetonitrile gradient program was established for simultaneous quantification of shikimate-derived secondary metabolites in cultured cells of Lithospermum erythrorhizon. The cells cultured in pigment production medium (M-9) are capable of producing five highly hydrophilic compounds such as p-hydroxybenzoic acid-O-glucoside and lithospermic acid B, as well as eleven lipophilic compounds including echinofuran B and acetylshikonin. In addition to the wide polarities of those compounds, many of them are unstable under light, dryness, oxygen and heating. Thus, a new extraction procedure for all these compounds was also established by use of ultrasonication under ice-water chilling with MeOH as the solvent. This procedure was applied to the quantitative analyses of these compounds in cell cultures and hairy root cultures of Lithospermum, and in the intact plants as well.

Topics: Anthraquinones; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Benzofurans; Cells, Cultured; Chromatography, High Pressure Liquid; Cold Temperature; Depsides; Furans; Glucosides; Naphthoquinones; Parabens; Plant Roots; Plants, Medicinal; Shikimic Acid; Solvents; Sonication

The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1). Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.

Topics: Alkyl and Aryl Transferases; Anti-Infective Agents; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Genes, Bacterial; Genetic Engineering; Magnoliopsida; Naphthoquinones; Plant Roots; Plants, Genetically Modified; Plants, Medicinal; Transformation, Genetic

beta-Alkannin (shikonin), a compound isolated from the root of Lithospermum erythrorhizon Siebold Zucc., has been used as a purple dye in ancient Japan and is known to exert an anti-inflammatory activity. This study aimed to understand the biological activity in terms of physico-chemical characteristics of beta-alkannin. Several physico-chemical properties including proton dissociation constants, half-wave potentials and molecular orbital energy of beta-alkannin were elucidated. This compound shows highly efficient antioxidative activities against several types of reactive oxygen species (ROS), such as singlet oxygen ((1)O2). superoxide anion radical (.O2), hydroxyl radical (.OH) and tert-butyl peroxyl radical (BuOO.) as well as iron-dependent microsomal lipid peroxidation. During the reactions of beta-alkannin with 1O2, .O2- and BuOO., intermediate organic radicals due to beta-alkannin were detectable by ESR spectrometry. Compared with the radicals due to naphthazarin, the structural skeleton of beta-alkannin, the beta-alkannin radical observed as an intermediate in the reactions with (1)O2, and .O2- was concluded to be a semiquinone radical. On the other hand, during the reactions of beta-alkannin and naphthazarin with BuOO., ESR spectra different from the semiquinone radical were observed, and proposed to result from the abstraction of hydrogen atoms from phenolic hydroxyl groups of beta-alkannin by BuOO.. Based on the ROS-scavenging abilities of beta-alkannin, the compound was concluded to react directly with ROS and exhibits antioxidative activity, which in turn exerts anti-inflammatory activity.

Topics: Acetonitriles; Anti-Inflammatory Agents, Non-Steroidal; Deuterium Oxide; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Lipid Peroxidation; Molecular Structure; Naphthoquinones; Peroxides; Plants, Medicinal; Potentiometry; Reactive Oxygen Species; Spin Trapping

beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermium radix, inhibited the growth of various lines of cancer cells derived from human solid tumors at low concentrations between 10(-8) and 10(-6) M. When HL-60 cells were treated with 10(-6) M beta-HIVS for 3 h, characteristic features of apoptosis, such as DNA fragmentation, nuclear fragmentation, and activation of caspase-3-like activity, were observed. The most characteristic features of the effect of beta-HIVS were the remarkable morphological changes induced upon treatment of HL-60 cells with beta-HIVS, as visualized on the staining of actin filaments with phalloidin labeled with tetramethylrhodamine B isothiocyanate. Moreover, activation of MAP kinases, such as ERK2, JNK and p38, was detected after treatment with 10(-6) M beta-HIVS preceding the appearance of the characteristics of apoptosis, and the features of the activation of these MAP kinases were quite different from those of Fas and anticancer drug-induced apoptosis. The activation of JNK by beta-HIVS was not inhibited by inhibitors of caspases, suggesting that JNK is located either upstream or independent of the caspase signaling pathway. beta-HIVS did not inhibit the activity of topoisomerase II. These results indicate that beta-HIVS induces apoptosis in HL-60 cells through a mechanism unlike those reported for anti-Fas antibodies and etoposide.

Topics: Antineoplastic Agents; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Caspase 3; Caspases; Cell Division; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Etoposide; fas Receptor; HL-60 Cells; Humans; Naphthoquinones; Nucleic Acid Synthesis Inhibitors; Signal Transduction; Tumor Cells, Cultured

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation. The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.

Topics: Cell Line, Transformed; Escherichia coli; Genetic Engineering; Genetic Vectors; Kinetics; Naphthoquinones; Oxo-Acid-Lyases; Plant Roots; Plants; Recombinant Proteins; Rhizobium; Shikimic Acid; Transfection

Shikonin is one of the active components isolated from the root of Arnebia euchrona (Royle) Johnst. It has been shown to possess significant antibacterial, antiinflammatory and antitumour activities and has been used clinically. In this paper, rat liver microsomes were incubated in vitro to study the metabolism of shikonin and a reverse-phase high performance liquid chromatography/photodiode array detector (DAD) method was used to separate and detect the metabolites. The effects of several factors on the metabolism were investigated and the metabolic system was optimized. The main metabolites were purified by RP-HPLC and their structures were determined by UV, 1H-NMR and MS. S-1 was dihydroxylated shikonin, S-2 was 2-OH shikonin and S-3 was 6-OH or 7-OH shikonin.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Chromatography, High Pressure Liquid; Magnetic Resonance Spectroscopy; Male; Mass Spectrometry; Microsomes, Liver; Molecular Structure; Naphthoquinones; Rats; Rats, Wistar; Spectrophotometry, Ultraviolet

Apoptosis is a new therapeutic target of cancer research. Shikonin isolated from Lithospermum erythrorhizon, a traditional oriental medicinal herb, was observed to induce apoptosis in HL60 human premyelocytic leukemia cell line. Shikonin induced DNA fragmentation into the multiples of 180 bp and increased the percentage of hypodiploid cells in flow cytometry after propidium iodide staining. The increase of apoptotic cells was preceded by the activation of caspase-3, which was reported to play a central role in apoptotic process. The DNA fragmentation induced by shikonin was completely inhibited by the pretreatment of z-VAD-fmk, a specific inhibitor of caspase, clearly showing that the mode of cell death is apoptotic.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspases; HL-60 Cells; Humans; Korea; Medicine, East Asian Traditional; Naphthoquinones; Plants, Medicinal; Poly(ADP-ribose) Polymerases

Five red shikonin pigments, deoxyshikonin, shikonin, acetylshikonin, isobutylshikonin, and beta-hydroxyisovalerylshikonin, were isolated from the roots of Lithospermum erythrorhizon cultivated in Korea. The purified pigments were red, purple, and blue at acidic, neutral, and alkaline pH values, respectively. Physical stability of the purified pigments against heat and light in an aqueous solution was examined for possible value-added food colorants. The thermal degradation reactions were carried out at pH 3.0 (50 mM glycine buffer) in 50% EtOH/H(2)O. Deoxyshikonin (t(1/2) = 14.6 h, 60 degrees C) and isobutylshikinin (t(1/2) = 19.3 h, 60 degrees C) are relatively less stable than other shikonin derivatives (t(1/2) = 40-50 h, 60 degrees C). Activation energies of thermal degradation of the isolated pigments were calculated. The activation energy of deoxyshikonin was the highest (12.5 kcal mol(-)(1)) and that of beta-hydroxyisovalerylshikonin was the lowest (1.71 kcal mol(-)(1)) value. Light stabilities of the pigments were similar to each other in that the half-life values of photodegradation for 20000 lx light intensity were 4.2-5.1 h.

Topics: Drug Stability; Humans; Hydrogen-Ion Concentration; Korea; Naphthoquinones; Pigments, Biological; Plant Roots; Plants, Medicinal

The carbon skeleton of the naphthoquinone pigment shikonin, which is produced in Lithospermum erythrorhizon Sieb. et Zucc. cell-suspension cultures, is partly derived from the isoprenoid biosynthetic pathway. The requirement of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34), a key enzyme of the mevalonate route to isoprenoids, for shikonin synthesis was investigated. Conserved regions of sequences from plant HMGR genes were used to design polymerase chain reaction (PCR) primers for the cloning of a cDNA fragment from L. erythrorhizon. The resulting 443-bp clone was used as a probe for Northern analyses and hybridized to an mRNA of approx. 2.5 kb. Under shikonin-producing conditions, microsomal HMGR enzyme activity as well as mRNA level closely correlated with the accumulation of shikonin derivatives. White light, which inhibits shikonin formation, was shown to strongly suppress HMGR gene expression. The results presented here indicate that HMGR plays a significant role in the regulation of shikonin biosynthesis and that the control appears to act at the transcriptional level.

Topics: Acyl Coenzyme A; Amino Acid Sequence; Cells, Cultured; Microsomes; Molecular Sequence Data; Naphthoquinones; Plants; Sequence Homology, Amino Acid

The present study was carried out to compare the accelerating effect of shikonin and alkannin and to elucidate the expression of CD antigen and histological changes on the proliferation of granulation tissue in rats. Shikonin and alkannin produced a dose-dependent acceleration of the cotton pellet-induced granuloma formation and this accelerating potency of both compounds on the proliferation of granulation tissue was about the same 5 and 10 d after implantation of the cotton pellet. Also, both compounds increased the ratio of CD11b+ cells in the granulation tissue 5 and 10 d after implantation of the cotton pellet. Both compounds increased the expression of CD11b+ cells with granulocytes such as macrophages and histiocytes, and then accelerated the proliferation of fibroblasts and collagen fiber. On the other hand, neither compound increased the ratio of CD3+ cells in the granulation tissue after 5 and 10 d. These results suggest that shikonin and alkannin accelerate the proliferation of granulation tissue induced by the cotton pellet and this accelerating effect may be attributed to an increase in the expression of CD11b+ cells, and the acceleration of the proliferation of fibroblasts and collagen fiber in the granulation tissue.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; CD3 Complex; Flow Cytometry; Gossypium; Granulation Tissue; Granuloma, Foreign-Body; Lymphocytes; Macrophage-1 Antigen; Male; Naphthoquinones; Rats; Rats, Wistar; Skin; Stimulation, Chemical

The effect of shikonin (SK) on granulation tissue formation was biochemically evaluated and the biological effect of SK on cotton pellet induced granulation tissue formation and the induction of hind paw edema was compared with that of lambda-carrageenan (carrageenan) in rats. The dry weight of granulation tissue formed was increased by SK. The amounts of hemoglobin and hydroxyproline in granulation tissue were also increased by SK. These results suggest that SK has an enhancing effect on the formation of granulation tissue, accompanied by the proliferation of capillaries and the increased production of collagen in rats. SK showed mild stimulation toward swelling when injected into the hind paws of rats, as did carrageenan, while it showed a marked enhancing effect on granulation tissue formation compared with carrageenan. These results suggest that the mechanism underlying the biological effect of SK on granulation tissue formation is different from that of carrageenan, though the mild stimulation by SK might still contribute in part to the enhancing effect on granulation tissue formation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Edema; Excipients; Granulation Tissue; Hindlimb; Male; Naphthoquinones; Rats; Rats, Wistar

An HPLC method was developed to resolve the enantiomers of shikonin derivatives of the Lithospermi Radix. The optimum mobile phase on a Chiracel AD column was 5% isopropanol in n-hexane with flow rate of 1 ml/min. Establishment of this method made possible to determine the ratios of shikonin/acetylshikonin or alkanin/acetylalkanin in the same root. The correlation of the ratios of these substance pairs appeared characteristic for the country where they were originated from. All of the Korean species showed significantly higher ratios of shikonin/acetylshikonin and alkanin/acetylalkanin than the Chinese ones. This method would be useful to determine the origin of Lithospermi Radix.

Topics: 2-Propanol; Acetylation; Anti-Inflammatory Agents, Non-Steroidal; Calibration; Chromatography, High Pressure Liquid; Korea; Naphthoquinones; Plant Roots; Plants, Medicinal; Solvents; Stereoisomerism

The effects of shikonnin (SK) and its optical isomer alkannin (AK) on the hydroxyl radical (HO.) generation system including iron ions were evaluated using the spin trap method by ESR spectroscopy. 5,5-Dimethyl-1-pyrroline-1-oxide (DMPO) was used as a spin trap agent and HO. was generated by a reaction between an iron ion and hydrogen peroxide, which is called Fenton reaction system. SK inhibited the HO. spin adduct (DMPO-OH) yielded in a dose-dependent manner. In this effect no difference was observed between SK and AK. When different concentrations of DMPO were used for the confirmation of its competitive reaction, no difference was also observed in the concentration of SK required to reduce the amount of the DMPO-OH by 50% (ID50). These findings suggested that the inhibitory effect of SK against the thus yielded DMPO-OH was not generated by the scavenging for HO., but by the inhibition on the Fenton reaction system. The mechanism of the inhibition on this system may be based on the formation of a complex between SK and the iron ion. The molar ratio of SK to the iron ion in the complex was considered 2 to 1 (2:1), because the concentrations of the observed ID50 and the used iron ion exhibited the same value. In addition, the same result was also obtained from the study using spectroscopic analysis.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Cyclic N-Oxides; Depression, Chemical; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Hydrogen Peroxide; Hydroxyl Radical; Iron; Isomerism; Naphthoquinones; Spin Trapping

Two cDNA clones (LEPAL-1 and LEPAL-2) encoding phenylalanine ammonia-lyase were isolated from cell suspension cultures of Lithospermum erythrorhizon. Northern kinetic studies showed that LEPAL-1 mRNA contents markedly increased one day after inoculation of the cells into fresh medium, then decreased to the steady-state level. The course of mRNA accumulation paralleled that of PAL enzyme activity. The rapid induction of PAL activity seems to reflect the induction of dihydroechinofuran biosynthesis, while shikonin was produced at the steady-state level of PAL activity. The course of LEPAL-2 mRNA accumulation seemed to be similar to, but much lower than that of LEPAL-1. In the intact plant, both genes are expressed mainly in the root, the organ in which shikonin is exclusively produced and accumulated. Genomic Southern blot analyses showed that both genes are present in the genome as single copies.

Topics: Amino Acid Sequence; Asia; Base Sequence; Biological Evolution; Cloning, Molecular; DNA, Complementary; Escherichia coli; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Naphthoquinones; Phenylalanine Ammonia-Lyase; Plant Proteins; Plants, Medicinal; RNA, Messenger; Sequence Alignment

Acetylshikonin, teracrylshikonin, beta,beta-dimethylacrylshikonin and shikonin, isolated from Arnebia euchroma, inhibited collagen (10 micrograms/ml)-induced aggregation of washed rabbit platelets in a concentration-dependent manner with IC50 values of 2.1 +/- 0.2, 2.8 +/- 0.3, 4.2 +/- 0.5 and 10.7 +/- 0.7 microM, respectively. Acetylshikonin also inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA, 100 microM), U46619 (1 microM), platelet-activating factor (PAF, 3.6 nM) and thrombin (0.1 U/ml) in a concentration-dependent manner. The IC50 values of acetylshikonin on the inhibition of these four agonists-induced platelet aggregation were 3.1 +/- 0.4, 2.2 +/- 0.2, 8.0 +/- 0.6 and 12.7 +/- 1.0 microM, respectively. The thromboxane B2 formation caused by collagen, PAF and thrombin was inhibited by acetylshikonin, while formations of thromboxane B2 and prostaglandin D2 caused by AA were not inhibited. Acetylshikonin did not inhibit cyclooxygenase activity since it did not attenuate prostaglandin E2 formation after incubation of sheep vesicular gland microsomes with AA. Acetylshikonin suppressed both the rise of intracellular Ca2+ concentration and the generation of [3H]inositol monophosphate caused by these five aggregation inducers. Platelet cyclic AMP level was unaffected by acetylshikonin. These data indicate that acetylshikonin inhibits platelet activation by suppression of phosphoinositide breakdown.

Topics: Animals; Anthraquinones; Blood Platelets; Calcium; Collagen; Cyclic AMP; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Naphthoquinones; Phosphatidylinositols; Plants, Medicinal; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Rabbits; Thromboxane B2

Compounds bearing an acyl group of a various size at 1'-OH of shikonin were synthesized as acyl analogues of shikonin, which was isolated from the root of Lithospermum erythrorhizon, and evaluated for inhibitory effect on topoisomerase-I activity. A selective acylation at 1'-OH of shikonin in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine gave rise to a good yield of corresponding acylshikonin derivatives. In general, analogues with an acyl group of shorter chain lengths (C2-C6) exerted a stronger inhibitory action than those with longer chain lengths (C7-C20). While the halogen substitution at C-2 of the acetyl moiety failed to increase the inhibitory potency, the placement of double bonds in the acyl group (C5-C7) augmented the potency remarkably. Of the 32 derivatives evaluated, 15 compounds exhibited a higher inhibitory effect than shikonin. Noteworthy, the inhibitory potency of acetylshikonin, propanoylshikonin, and 4-pentenoylshikonin was approximately 4-fold greater than that of camptothecin. All these data suggest that the size of acyl moiety is important for the enhancement of potency, and the presence of olefinic double bonds is also beneficial.

Topics: Acetylation; Antineoplastic Agents, Phytogenic; DNA Topoisomerases, Type I; DNA, Superhelical; HeLa Cells; Humans; Naphthoquinones; Plant Extracts; Plant Roots; Topoisomerase I Inhibitors

The discoloration of shikonin (1) and beta-carotene (2), occurring during storage of their ethanol solutions in the presence of oxygen in an illuminated room, was remarkably suppressed by hydrolyzable tannins, such as geraniin (4) and tannic acid JP (3) in the solution. The inhibitory effect of tannins was enhanced by the coexistence of metallic ion. The irradiation with ultraviolet lamp (254 and 365 nm) gave, at the first stage of the discoloration, two products, one of which was found to be 5,8-dihydroxy-2-(1-hydroxy-3-oxo-4-methyl-4-pentenyl)-1,4-naphthoquinone (7). The presence of hydrolyzable tannins induced higher accumulation of these two products in the solution, showing that the secondary structural transformations of these two products were strongly inhibited by these coexisting tannins. These results suggest that tannins could be efficient inhibitors of discoloration of natural pigments.

Topics: beta Carotene; Carotenoids; Drug Interactions; Drug Stability; Naphthoquinones; Photolysis; Solutions; Tannins; Ultraviolet Rays

The present study was carried out to compare the accelerative effect of shikonin (R-type), alkannin (S-type), and acetylshikonin on the proliferation of granulation tissue in rats, and to elucidate the correlation between the potency of the effect and their optical activity. Koushikon mainly contained the R-type of acetylshikonin, and Nanshikon mainly contained the S-type of acetylshikonin. Each compound produced a dose-dependent acceleration of the cotton pellet-induced granuloma formation. In comparing identical doses of shikonin, alkannin and acetylshikonin, the potency of their accelerative effects on the proliferation of granulation tissue was about the same. This result suggests that their absolute configurations (R-type or S-type) and their acetylation on the hydroxy group of the sidechain of shikonin or alkannin may not be important in producing the effect.

Topics: Animals; Anthraquinones; Anti-Inflammatory Agents, Non-Steroidal; Cell Division; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Gossypium; Granulation Tissue; Isomerism; Male; Naphthoquinones; Rats; Rats, Wistar

Effects of in situ extraction, fungal elicitation, a permeabilizing agent, and the oxygen transfer rate on shikonin production in transformed suspension and hairy root cultures of Lithospermum erythrorhizon were studied. Shikonin production with in situ extraction in transformed cell and hairy root cultures by n-hexadecane was 7.6 and 3 times higher than those of the control culture. Shikonin production of transformed L. erythrohizon increased with the enhanced gas exchange, and in situ extraction also increased sucrose consumption and shikonin production. The optimal volume of n-hexadecane in the hairy root culture was similar to that in the transformed cell cultures. In situ extraction at an earlier stage significantly enhanced shikonin production both in transformed cell and hairy root cultures. Dimethylsulfoxide used as a permeabilizing agent was harmful to cell growth and shikonin production, and permeabilizing was unnecessary when in situ extraction was applied. This occurred because with the solvent addition, most shikonin was spontaneously released from the cells and was dissolved in the solvent layer. The combined addition of n-hexadecane of the extract of the fungus Penicillium as an elicitor seemed to result in a higher production of shikonin both in cell suspensions and transformed root cultures. However, an increase of shikonin induction by fungal elicitation in a hairy root culture was not significant in comparison with that of normal cell cultures.

Topics: Agrobacterium tumefaciens; Alkanes; Cells, Cultured; Gene Expression Regulation, Plant; Genetic Vectors; Glucose; Naphthoquinones; Oxygen; Penicillium; Plant Cells; Plant Extracts; Plant Tumors; Plants; Transformation, Genetic

Shikonin is one of the active components isolated from the dry root of Arnebia euchroma (Royle) Johnst (AERJ). It has been shown to have anti inflammatory activity on formaldehyde induced paw swelling in rats. Preparations of AERJ has been used clinically in curing phlebitis and vascular purpura. In the present study, sc administered shikonin was shown to have significant inhibitory effects on ear edema induced by croton oil in mice and paw swelling induced by yeast in rats. In order to investigate the influence of shikonin on biosynthesis of LTB4 and 5-HETE, an in vitro leukocyte incubation system was adopted. The results showed that shikonin has fairly strong inhibitory effects on LTB4 and 5-HETE biosynthesis. Its effects and concentrations fit a positive relationship within the range of 10(-7)-10(-4) mol.L-1. The equations of inhibition (Y) versus concentration (X, LogC) obtained by linear regression were Y = 166 + 18.7X (r = 0.9319) for LTB4 and Y = 173 + 18.7X (r = 0.9856) for 5-HETE. The IC50 were 6.2 x 10(-7) and 2.6 x 10(-7) mol.L-1, respectively. The results also indicate that natural shikonin derivatives have similar inhibitory effects on LTB4 biosynthesis. These results suggest that inhibition of LTB4 and 5-HETE may play a major role in the mechanism of anti inflammatory effects of shikonin.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Croton Oil; Edema; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Mice; Naphthoquinones; Rats; Rats, Wistar

The circular dichroism (CD) and electronic absorption spectra of seven derivatives of shikonin and alkannin isolated from the roots of Onosma confertum W.W. Smith (from Sichuan) and Arnebia euchroma (Royle) Johust (from Xinjiang) were described. Each pair of CD spectra obtained was exact mirror images and the UV spectra of these compounds were very similar. In comparison with the CD spectra of shikonin given in literature, the configuration of the pigments from Sichuan was assigned R and from Xinjiang were all S.

Topics: Circular Dichroism; Drugs, Chinese Herbal; Molecular Conformation; Naphthoquinones; Species Specificity; Stereoisomerism

Four antiplatelet components were isolated from Arnebia euchroma. They inhibited the aggregation of washed rabbit platelets caused by collagen, arachidonic acid, platelet-activating factor, ADP, or thrombin. The potency of inhibiting collagen-induced platelet aggregation is in the following order: acetylshikonin (IC50 = 2.1 microM), teracrylshikonin (2.8 microM), beta, beta-dimethylacrylshikonin (4.2 microM), and shikonin (10.7 microM). In rat aorta, acetylshikonin and shikonin inhibited high potassium and norepinephrine-induced contractions, while beta, beta-dimethylacrylshikonin and teracrylshikonin potentiated the phasic contraction caused by norepinephrine.

Topics: Animals; Drugs, Chinese Herbal; Female; In Vitro Techniques; Male; Molecular Structure; Naphthoquinones; Platelet Aggregation Inhibitors; Rabbits; Rats; Rats, Wistar

This study was carried out to compare the accelerative effect in ether extracts of "Koushikon" and "Nanshikon" on proliferation of granuloma tissue in rats, and to elucidate this effect on optical isomer of naphthoquinone derivatives in those extracts. The content of total naphthoquinone derivatives in the ether extracts of Koushikon and Nanshikon were found to be 56.1% and 25.4%. Among naphthoquinone derivatives, Koushikon contained mostly acetyl derivative and Nanshikon mostly teracryl derivative. The percentage of R-type (shikonin-type) in total naphthoquinone derivatives of the extracts was 85.5% and 3.8%. Each ether extract showed a dose-dependent acceleration on the cotton pellet-induced granuloma formation. Comparison with corresponding doses containing the same quantity of naphthoquinone derivatives showed the accelerative potency of ether extracts of Koushikon and Nanshikon to be about the same. The result suggests that the accelerative effect on proliferation of granuloma tissue depends primarily on the total content of naphthoquinone derivatives, and not on the ratio of the optically active isomers.

Topics: Animals; Cell Division; Drugs, Chinese Herbal; Granuloma; Male; Naphthoquinones; Rats; Rats, Wistar; Stereoisomerism

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

Of twelve reduced and acetylated derivatives of shikonin, a chemical constituent of Shikon, the accelerating activity on granuloma formation and the inhibitory activity on delayed-type allergy were investigated in order to find a compound having more characteristic effect than shikonin on wound healing in experimental animals. As a result, it was found that a reduced and pentaacetylated derivative of shikonin, MDS-004, has more excellent pharmacological activity. MDS-004 (0.1-1 mg/pellet) accelerated dose-dependently felt-pellet-induced granuloma formation when given topically together with felt-pellets in rats. It also produced strong inhibition against delayed-type allergies (ear edema) caused by oxazolone and dinitrofluorobenzene by topical application of up to 1 mg/ear to the ear skin of mice; its potency was far superior to that of shikonin. Orally administered MDS-004, unlike shikonin, inhibited carrageenan-induced hind paw edema, and exhibited tendency to heal acetic acid-induced gastric ulcer in rats. However, MDS-004, as well as commercial wound healing drugs tested and shikonin, did not show any healing action in the incised and open wound models in rats, if applied topically to the wound as 5 and 10% powders. On the other hand, MDS-004 did not produce irritative action on the ear skin at a topical dose of 1 mg/ear different from shikonin, and any behavioral changes after oral administration of 100 mg/kg in mice. These results suggest that a white powder MDS-004, different from deep purple shikonin, has accelerating action on granuloma formation without irritative action and stronger inhibitory action on delayed-type allergy by topical application than shikonin.

Topics: Animals; Granulation Tissue; Granuloma; Hypersensitivity, Delayed; Male; Mice; Mice, Inbred Strains; Naphthoquinones; Rats; Rats, Inbred Strains; Wound Healing

Topics: Acoustics; Animals; Humans; Lasers; Mice; Mice, Hairless; Naphthoquinones; Photometry; Skin Absorption

Modifying effects of a fungal product, flavoglaucin, and four plant-derived chemicals, shikonin, gingerol, oleanolic acid and paeoniflorin, on intestinal carcinogenesis were examined in a rat model using azoxymethane (AOM). A total of 280 male F344 rats, 6 weeks old, were divided into 12 groups. Group 1 (30 rats) was given two subcutaneous injections of 15 mg/kg of AOM at the start of the experiment. Groups 2 (30 rats), 3 (20 rats), 4 (20 rats), 5 (30 rats) and 6 (30 rats) received a test chemical (flavoglaucin, shikonin, gingerol, oleanolic acid or paeoniflorin, respectively) in the diet at a concentration of 0.02% for 3 weeks, during which time AOM was applied, and then kept on basal diet until the end of experiment (one year). Groups 7-11 (each 20 rats) were given a test chemical corresponding to Groups 2-6, respectively. Group 12 (20 rats) served as a control. The incidence and average number of intestinal tumors in Group 2 (47%, 0.57 +/- 0.68) were significantly less than in Group 1 (74%, 1.07 +/- 0.87) (P < 0.05, respectively). Multiplicity of intestinal neoplasms of Group 3 (0.55 +/- 0.60) or 4 (0.47 +/- 0.51) was also significantly smaller than that of Group 1 (P < 0.05 and P < 0.01, respectively). These results suggest that flavoglaucin, shikonin and gingerol might be promising chemopreventive agents for intestinal neoplasia.

Topics: Animals; Antineoplastic Agents, Phytogenic; Azoxymethane; Benzoates; Bridged-Ring Compounds; Catechols; Drug Screening Assays, Antitumor; Fatty Alcohols; Gentisates; Glucosides; Intestinal Neoplasms; Male; Monoterpenes; Mutagens; Naphthoquinones; Oleanolic Acid; Rats; Rats, Inbred F344

Roots, root cortices and root corks from 6 species of Boraginaceae are used as zicao in commercial crude drugs. This paper reports the determination of naphthaquinone pigments (such as total pigments, beta, beta-dimethylacryshikonin, acetylshikonin and shikonin) in 12 samples of 6 plants. The quality of various drugs was evaluated by determining the contents of the above principles.

Topics: Anthraquinones; Antineoplastic Agents, Phytogenic; Chromatography, Thin Layer; Densitometry; Drugs, Chinese Herbal; Naphthoquinones; Quality Control

The shikonin mixture was used for 19 cases of later-stage lung cancer who were not the candidates for operation, radiotherapy and chemotherapy. The clinical observation showed that shikonin mixture could inhibit the growth of lung cancer and improve the immune function of the body. The tumors were reduced over 25% in diameter. The effective rate was 63.3%, remission rate 36.9%, survival rate of one year 47.3%. The intermedium survival period was about 10 months, including adenocarcinoma 10 months, squamous carcinoma 12 months. After treatment the life quality of patients were greatly improved. The patients got better appetite and their body weights were increased. They could manage themselves in daily life. The Karnofsky scores were enhanced by 20. The authors also observed that shikonin mixture could relieve such symptoms as cough, bloody sputum and chest pain caused by lung cancer. The levels of cells and interleukin-2 were increased (P less than 0.001). It had no harmful effects on peripheral blood picture, heart, kidney and liver. Shikonin mixture is safe and effective for later-stage cancer.

Topics: Adenocarcinoma; Adult; Antineoplastic Agents, Phytogenic; Carcinoma, Bronchogenic; Carcinoma, Squamous Cell; Female; Ginsenosides; Humans; Interleukin-2; Killer Cells, Natural; Lung Neoplasms; Male; Middle Aged; Naphthoquinones; Saponins

Naphthoquinone pigment-LIII, an extract from Arnebia euchroma, could apparently inhibit the proliferation of stomach cancer cell line and esophagus cancer cell line. At the effective concentration of 5 micrograms/ml, the mitotic index and growth curve declined without showing any damage to human normal cells. At 5-10 micrograms/ml, the colony efficiency of cancer cells became significantly low. The anti-cancer effect of Naphthoquinone pigment-LIII might be related to its role of influencing the amount of RNA and ultrastructure of cancer cells which was discussed in this paper.

Topics: Antineoplastic Agents, Phytogenic; Esophageal Neoplasms; Humans; Naphthoquinones; Stomach Neoplasms; Tumor Cells, Cultured

A novel in vitro percutaneous absorptiometry utilizing a portable open-ended photoacoustic (PA) cell as the longitudinal diffusion cell was developed. With this system it was feasible to measure the reduction of drug applied to a membrane by the PA method and the amount of drug penetrating the membrane to the diffusion cell by absorbance, simultaneously in real time. A shikonin ointment prepared in a hydrocarbon vehicle was used as the model sample. The in vitro percutaneous absorptiometry was performed by means of a physiological saline solution and the skin of a hairless mouse. As a result, after the lag time the absorbance increased in proportion to time, whereas the PA signal reduced in proportion to the square root of time. As the signal obtained by the PA method corresponds to the amount of drug released from the ointment, a good correlation with Higuchi's theory is attained. Consequently, these results suggested the usefulness of this novel percutaneous absorptiometry technique which utilizes the PA method.

Topics: Absorption; Acoustics; Animals; Antineoplastic Agents, Phytogenic; Cell Membrane Permeability; Male; Membranes, Artificial; Mice; Mice, Hairless; Naphthoquinones; Ointments; Photometry; Skin

The present study was carried out to evaluate an equivalence of pharmacological properties between natural crude drugs and their cultured cells. The effects of ether extract of Lithospermi Radix and cultured cells of Lithospermum erythrorhizon Sieb. et Zucc. and aqueous extract of Coptidis Rizoma and cultured cells of Coptis japonica Makino var. dissecta Nakai on proliferation of granulation tissue in rats were compared. The ether extracts of Lithospermi Radix and the cultured cells enhanced proliferation of granulation tissue by the cotton pellet method. The potency of both extract was about the same, if results were compared with the corresponding doses which contained the same quantity of shikonin derivatives. On the other hand, the aqueous extracts of Coptidis Rhizoma and the cultured cells inhibited it. The potency of both extract was about the same, if results were compared with the corresponding doses which contained the same quantity of berberine-type alkaloids. From these results, to evaluate an equivalence of pharmacological properties between natural crude drugs and their cultured cells, it is concluded that their qualities and quantities are not so different each other and the almost same pharmacological effect expected on the basis of their uses is required.

Topics: Alkaloids; Animals; Antineoplastic Agents, Phytogenic; Cell Division; Cells, Cultured; Drugs, Chinese Herbal; Granulation Tissue; Male; Naphthoquinones; Plants, Medicinal; Rats; Rats, Inbred Strains

A prenyltransferase (EC 2.5.1.1) was isolated from cell cultures of Lithospermum erythrorhizon. The enzyme was purified 92-fold by subsequent chromatography on DEAE-Sephacel, phenyl-Sepharose, and Sephadex G-150. Geranyl pyrophosphate (GPP) was the sole product of the enzymatic reaction with dimethylallyl pyrophosphate and isopentenyl pyrophosphate as the substrates. The enzyme showed a molecular weight of 73,000, estimated by gel chromatography on Sephadex G-150, and an isoelectric point at pH 4.95, determined by analytical isoelectric focusing. It had an absolute requirement for a divalent cation with Mg2+ and Mn2+ being most effective. The enzyme was soluble rather than membrane-bound. The physiological role of this prenyltransferase probably is to supply GPP for the biosynthesis of shikonin. It is the first chain-length specific geranyl pyrophosphate synthase reported from eukaryotic cells.

Topics: Chemical Phenomena; Chemistry; Chromatography, DEAE-Cellulose; Chromatography, Gas; Dimethylallyltranstransferase; Fluorides; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Magnesium; Manganese; Molecular Weight; Naphthoquinones; Plants, Medicinal; Transferases

Topics: Absorption; Animals; Male; Mice; Naphthoquinones; Tissue Distribution

Topics: Chromatography, Thin Layer; Drugs, Chinese Herbal; Naphthoquinones; Pigments, Biological