ABSTRACT
Rhizoma polygonati (Huangjing, RP) has been used for a long history with many chemical components in inducing anti-cancer, anti-aging, anti-diabetes, anti-fatigue, and more prevention of diseases or acts as nutrition sources in food. Here we investigated RP extract combination with kinase inhibitors in anti-cell growth and blockade in pathways targeting kinases. Experimental investigation and network pharmacology analysis were applied to test the potent kinase-mediated signaling. Herbzyme activity was determined by substrate with optical density measurement. Extract of processed RP inhibits cell growth in a much greater manner than alone when applied in combination with inhibitors of mTOR or EGFR. Moreover, processing methods of RP from Mount Tai (RP-Mount Tai) play essential roles in herbzyme activity of phosphatase suggesting the interface is also essential, in addition to the chemical component. The network pharmacology analysis showed the chemical component and target networks involving AKT and mTOR, which is consistent with experimental validation. Finally, EGFR inhibitor could be associated with nano-extract of RP-Mount Tai but not significantly affects the phosphatase herbzyme activity in vitro. Thus the processed extract of RP-Mount Tai may play a dual role in the inhibition of cell proliferation signaling by both chemical component and nanoscale herbzyme of phosphatase activity to inhibit kinases including mTOR/AKT in potent drug delivery of kinase inhibitors.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Network Pharmacology/methods , Plant Extracts/pharmacology , Polygonatum , Cell Enlargement/drug effects , Cell Line, Tumor , Humans , Protein Interaction Maps , Proto-Oncogene Proteins c-akt/drug effects , TOR Serine-Threonine Kinases/drug effectsABSTRACT
Genistein, a naturally occurring phytoestrogen and a member of the large class of compounds known as isoflavones, exerts protective effects in several diseases. Recent studies indicate that genistein plays a critical role in controlling body weight, obesity-associated insulin resistance, and metabolic disorders, but its target organs in reversing obesity and related pathological conditions remain unclear. In this study, we showed that mice supplemented with 0.2% genistein in a high-fat diet for 12 weeks showed enhanced metabolic homeostasis, including reduced obesity, improved glucose uptake and insulin sensitivity, and alleviated hepatic steatosis. We also observed a beiging phenomenon in the white adipose tissue and reversal of brown adipose tissue whitening in these mice. These changes led to enhanced resistance to cold stress. Altogether, our data suggest that the improved metabolic profile in mice treated with genistein is likely a result of enhanced adipose tissue function.
Subject(s)
Adipose Tissue, Beige/drug effects , Adipose Tissue, Beige/metabolism , Cold-Shock Response/drug effects , Cold-Shock Response/physiology , Genistein/pharmacology , Adipocytes, White/cytology , Adipocytes, White/drug effects , Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/drug effects , Adipose Tissue, White/metabolism , Animals , Body Weight/drug effects , Cell Enlargement/drug effects , Diet, High-Fat/adverse effects , Eating/drug effects , Energy Metabolism/drug effects , Insulin Resistance/physiology , Male , Mice , Mice, Inbred C57BL , Obesity/drug therapy , Obesity/metabolism , Obesity/pathology , Phytoestrogens/pharmacology , Protective Agents/pharmacologyABSTRACT
As a traditional plant medicine in tropical areas, Swietenia macrophylla seeds are usually applied for some chronic diseases, including hypertension, diabetes, and so on. Few studies have been carried out to identify the effective elements in seed extract and their indications. In this study, we first investigated the functions of the swietenine, an extract from S. macrophylla seeds, using a model of myocardial hypertrophy induced by isoprenaline (ISO). At cellular level, H9c2 cell hypertrophy was also established through the treatment with ISO. The cardiac pathological remodeling was evaluated by echocardiography and histological analysis. Western blot and RT-qPCR were used to detect the expression of possible hypertrophy-promoting genes. Here, our results indicated that swietenine remarkably attenuated ISO-induced myocardial hypertrophy in vivo and in vitro. Moreover, Akt phosphorylation, ANP and BNP mRNA expression were efficiently decreased. Based on these findings, we concluded that swietenine might be a promising anti-hypertrophic agent against cardiac hypertrophy.
Subject(s)
Cardiomegaly/prevention & control , Heart/drug effects , Limonins/pharmacology , Meliaceae/chemistry , Plant Extracts/pharmacology , Animals , Cardiomegaly/chemically induced , Cell Enlargement/drug effects , Cell Line , Cell Survival/drug effects , Isoproterenol/adverse effects , Limonins/isolation & purification , Male , Mice , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Organ Size/drug effects , Plant Extracts/isolation & purification , Rats , Seeds/chemistryABSTRACT
In this study, we used ICI 182 780 (ICI), an estrogen receptor (ER) antagonist, to investigate the estrogenic activity of Danshen, and to further explored whether Danshen extract can block Leu27IGF-II-induced hypertrophy in H9c2 cardiomyoblast cells. We first used an IGF-II analog Leu27IGF-II, which specifically activates IGF2R signaling cascades and induces H9c2 cardiomyoblast cell hypertrophy. However, Danshen extract completely inhibited Leu27IGF-II-induced cell size increase, ANP and BNP hypertrophic marker expression, and IGF2R induction. We also observed that Danshen extract inhibited calcineurin protein expression and NFAT3 nuclear translocation, leading to suppression of Leu27IGF-II-induced cardiac hypertrophy. Moreover, the anti-Leu27IGF-II-IGF2R signaling effect of Danshen was totally reversed by ICI, which suggest the cardio protective effect of Danshen is mediated through estrogen receptors. Our study suggests that, Danshen exerts estrogenic activity, and thus, it could be used as a selective ER modulator in IGFIIR induced hypertrophy model.
Subject(s)
Cell Enlargement/drug effects , Drugs, Chinese Herbal/pharmacology , Insulin-Like Growth Factor II/analogs & derivatives , Myoblasts, Cardiac/drug effects , Receptor, IGF Type 2/metabolism , Salvia miltiorrhiza/chemistry , Animals , Calcineurin/metabolism , Cardiomegaly/prevention & control , Cell Line , Cell Survival/drug effects , Drugs, Chinese Herbal/isolation & purification , Estrogen Receptor Antagonists/pharmacology , Fulvestrant/pharmacology , Insulin-Like Growth Factor II/pharmacology , Myoblasts, Cardiac/metabolism , Myoblasts, Cardiac/pathology , Protein Transport , Rats , Receptors, Estrogen/metabolism , Signal TransductionABSTRACT
KEY MESSAGE: Auxin treatment of grape (Vitis vinifera L.) berries delays ripening by inducing changes in gene expression and cell wall metabolism and could combat some deleterious climate change effects. Auxins are inhibitors of grape berry ripening and their application may be useful to delay harvest to counter effects of climate change. However, little is known about how this delay occurs. The expression of 1892 genes was significantly changed compared to the control during a 48 h time-course where the auxin 1-naphthaleneacetic acid (NAA) was applied to pre-veraison grape berries. Principal component analysis showed that the control and auxin-treated samples were most different at 3 h post-treatment when approximately three times more genes were induced than repressed by NAA. There was considerable cross-talk between hormone pathways, particularly between those of auxin and ethylene. Decreased expression of genes encoding putative cell wall catabolic enzymes (including those involved with pectin) and increased expression of putative cellulose synthases indicated that auxins may preserve cell wall structure. This was confirmed by immunochemical labelling of berry sections using antibodies that detect homogalacturonan (LM19) and methyl-esterified homogalacturonan (LM20) and by labelling with the CMB3a cellulose-binding module. Comparison of the auxin-induced changes in gene expression with the pattern of these genes during berry ripening showed that the effect on transcription is a mix of changes that may specifically alter the progress of berry development in a targeted manner and others that could be considered as non-specific changes. Several lines of evidence suggest that cell wall changes and associated berry softening are the first steps in ripening and that delaying cell expansion can delay ripening providing a possible mechanism for the observed auxin effects.
Subject(s)
Cell Wall/drug effects , Indoleacetic Acids/pharmacology , Plant Cells/drug effects , Plant Growth Regulators/pharmacology , Vitis/drug effects , Cell Enlargement/drug effects , Cell Wall/genetics , Fruit/drug effects , Fruit/growth & development , Gene Expression Regulation, Plant/drug effects , Naphthaleneacetic Acids/pharmacology , Plant Cells/physiology , Time , Vitis/growth & developmentABSTRACT
Through population-based studies, associations have been found between coffee drinking and numerous health benefits, including a reduced risk of cardiovascular disease. Active ingredients in coffee have therefore received considerable attention from researchers. A wide variety of effects have been attributed to cafestol, one of the major compounds in coffee beans. Because cardiac hypertrophy is an independent risk factor for cardiovascular events, this study examined whether cafestol inhibits urotensin II (U-II)-induced cardiomyocyte hypertrophy. Neonatal rat cardiomyocytes were exposed only to U-II (1 nM) or to U-II (1 nM) following 12-h pretreatment with cafestol (1-10 µ M). Cafestol (3-10 µ M) pretreatment significantly inhibited U-II-induced cardiomyocyte hypertrophy with an accompanying decrease in U-II-induced reactive oxygen species (ROS) production. Cafestol also inhibited U-II-induced phosphorylation of redox-sensitive extracellular signal-regulated kinase (ERK) and epidermal growth factor receptor transactivation. In addition, cafestol pretreatment increased Src homology region 2 domains-containing phosphatase-2 (SHP-2) activity, suggesting that cafestol prevents ROS-induced SHP-2 inactivation. Moreover, nuclear factor erythroid-2-related factor 2 (Nrf2) translocation and heme oxygenase-1 (HO-1) expression were enhanced by cafestol. Addition of brusatol (a specific inhibitor of Nrf2) or Nrf2 siRNA significantly attenuated cafestol-mediated inhibitory effects on U-II-stimulated ROS production and cardiomyocyte hypertrophy. In summary, our data indicate that cafestol prevented U-II-induced cardiomycyte hypertrophy through Nrf2/HO-1 activation and inhibition of redox signaling, resulting in cardioprotective effects. These novel findings suggest that cafestol could be applied in pharmacological therapy for cardiac diseases.
Subject(s)
Cell Enlargement/drug effects , Diterpenes/pharmacology , Myocytes, Cardiac/pathology , NF-E2-Related Factor 2/metabolism , Urotensins/adverse effects , Urotensins/antagonists & inhibitors , Animals , Cardiomegaly/drug therapy , Cells, Cultured , Depression, Chemical , Diterpenes/therapeutic use , ErbB Receptors/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Heme Oxygenase-1/metabolism , Phosphorylation/drug effects , Phytotherapy , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Rats , Reactive Oxygen Species/metabolism , Transcriptional Activation/drug effectsABSTRACT
Neurite outgrowth is crucial during neuronal development and regeneration, and strategies that aim at promoting neuritogenesis are beneficial for reconstructing synaptic connections after neuronal degeneration and injury. Using a bivalent analogue strategy as a successful approach, the current study identifies a series of novel dimeric securinine analogues as potent neurite outgrowth enhancers. Compounds 13, 14, 17-19, and 21-23, with different lengths of carbon chain of N,N-dialkyl substituting diacid amide linker between two securinine molecules at C-15 position, exhibited notable positive effects on both neuronal differentiation and neurite extension of neuronal cells. Compound 14, one of the most active compounds, was used as a representative compound for mechanistic studies. Its action on neurite outgrowth was through phosphorylation/activation of multiple signaling molecules including Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase (ERK) and Akt. These findings collectively identify a new group of beneficial compounds for neuritogenesis, and may provide insights on drug discovery of neural repair and regeneration.
Subject(s)
Azepines/chemical synthesis , Azepines/pharmacology , Cell Enlargement/drug effects , Heterocyclic Compounds, Bridged-Ring/chemical synthesis , Heterocyclic Compounds, Bridged-Ring/pharmacology , Neurites/drug effects , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/pharmacology , Animals , Azepines/chemistry , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Line, Tumor , Drug Design , Drug Evaluation, Preclinical , Extracellular Signal-Regulated MAP Kinases/metabolism , Heterocyclic Compounds, Bridged-Ring/chemistry , Immunohistochemistry , Lactones/chemistry , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mice , Molecular Structure , Neurites/physiology , Neuroprotective Agents/chemistry , Phosphorylation/drug effects , Piperidines/chemistry , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Prostaglandins (PGs) play important roles in diverse physiological processes in the central nervous system. PGD2 is the most abundant PG in the brain and acts through specific receptors, DP1 and CRTH2. We investigated the effects of PGD2 on the morphology of the hypothalamic cell line mHypoE-N37 (N37). In N37 cells, serum starvation induced neurite outgrowth and PGD2 elicited neurite retraction, although we failed to detect transcripts for DP1 and CRTH2. Such an effect of PGD2 was efficiently mimicked by its metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2. N-acetyl cysteine completely abolished the effect of PGD2, and reactive oxygen species (ROS) were considered to be important. Notably, neurite outgrowth was restored by PGD2 removal. These results suggest that PGD2 induces reversible neurite retraction in a ROS-mediated mechanism that does not involve any known receptor.
Subject(s)
Cell Enlargement/drug effects , Hypothalamus/cytology , Hypothalamus/metabolism , Neurites/physiology , Prostaglandin D2/administration & dosage , Reactive Oxygen Species/metabolism , Animals , Cell Line , Dose-Response Relationship, Drug , Hypothalamus/drug effects , Mice , Neurites/drug effects , Neurites/ultrastructureABSTRACT
Ever since the discovery of its neurite growth promoting activity in sympathetic and sensory ganglia, nerve growth factor (NGF) became the prototype of the large family of neurotrophins. The use of primary cultures and clonal cell lines has revealed several distinct actions of NGF and other neurotrophins. Among several models of NGF activity, the clonal cell line PC12 is the most widely employed. Thus, in the presence of NGF, through the activation of the transmembrane protein TrkA, these cells undergo a progressive mitotic arrest and start to grow electrically excitable neuritis. A vast number of studies opened intriguing aspects of NGF mechanisms of action, its biological properties, and potential use as therapeutic agents. In this context, identifying and utilizing small portions of NGF is of great interest and involves several human diseases including Alzheimer's disease. Here we report the specific action of the peptide encompassing the 1-14 sequence of the human NGF (NGF(1-14)), identified on the basis of scattered indications present in literature. The biological activity of NGF(1-14) was tested on PC12 cells, and its binding with TrkA was predicted by means of a computational approach. NGF(1-14) does not elicit the neurite outgrowth promoting activity, typical of the whole protein, and it only has a moderate action on PC12 proliferation. However, this peptide exerts, in a dose and time dependent fashion, an effective and specific NGF-like action on some highly conserved and biologically crucial intermediates of its intracellular targets such as Akt and CREB. These findings indicate that not all TrkA pathways must be at all times operative, and open the possibility of testing each of them in relation with specific NGF needs, biological actions, and potential therapeutic use.
Subject(s)
Nerve Growth Factor/chemistry , Nerve Growth Factor/pharmacology , Neuroprotective Agents/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Animals , Cell Enlargement/drug effects , Cell Proliferation/drug effects , Cyclic AMP Response Element-Binding Protein/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Humans , Molecular Docking Simulation , Nerve Growth Factor/genetics , Neurites/drug effects , Neurites/physiology , Neurogenesis/drug effects , Neuroprotective Agents/chemistry , PC12 Cells , Phosphorylation/drug effects , Rats , Receptor, trkA/metabolism , Time FactorsABSTRACT
RATIONALE: The excessive intake of vitamin A in the form of vitamin concentrate, supplement or vitamin-rich liver can result in hypervitaminosis A in man and animals. Although osteopathologies resulting from chronic vitamin A intoxication in cats are well characterized, no information is available concerning feline hypervitaminosis A-induced liver disease. CLINICAL SUMMARY: We report the first case of hepatic stellate cell lipidosis and hepatic fibrosis in a domestic cat that had been fed a diet based on raw beef liver. Radiographic examination revealed exostoses and ankylosis between vertebrae C1 and T7, compatible with deforming cervical spondylosis. Necropsy showed a slightly enlarged and light yellow to bronze liver. Microscopic and ultrastructural analyses of liver tissues revealed diffuse and severe liver fibrosis associated with hepatic stellate cell hyperplasia and hypertrophy. These cells showed immunopositive staining for α-smooth muscle actin and desmin markers. The necropsy findings of chronic liver disease coupled with osteopathology supported the diagnosis of hypervitaminosis A. PRACTICAL RELEVANCE: As in human hepatology, if there is dietary evidence to support increased intake of vitamin A, then hypervitaminosis A should be considered in the differential diagnosis of chronic liver disease in cats.
Subject(s)
Cat Diseases/diagnosis , Hypervitaminosis A/veterinary , Liver Cirrhosis/veterinary , Animals , Cat Diseases/diagnostic imaging , Cats , Cell Enlargement/drug effects , Hypervitaminosis A/chemically induced , Hypervitaminosis A/diagnostic imaging , Liver/drug effects , Liver Cirrhosis/chemically induced , Liver Cirrhosis/diagnostic imaging , Male , Radiography , Vitamin A/adverse effectsABSTRACT
BACKGROUND: Oxidized low density lipoprotein plays an important role in development of foam cells in atherosclerosis. The study was focused on regulation of primary human monocyte growth and CD11b expression in presence of Nigella sativa oil. METHODS: Primary human monocytes were isolated from whole blood and grown at 37°C and 5% CO2 saturation for five days prior to treatment with Nigella sativa oil. The cells were plated and washed before treatment with ox-LDL (10 µg/ml) as positive control and combined treatment of ox-LDL (10 µg/ml) and (140 ng/ml) Nigella sativa oil. The growth progression was monitored every 24 hours for 3 days. RESULTS: Macrophages showed reduced growth in comparison to monocytes 24 hours after treatment with Nigella sativa oil. The mean cell diameter was significantly different between untreated and treated condition in monocytes and macrophages (p < 0.001). Similarly, intracellular lipid accumulation was hindered in combined treatment with Nigella sativa oil. This was further supported by cell surface expression analysis, where CD11b was markedly reduced in cells treated with combination oxLDL and Nigella sativa oil compared to oxLDL alone. More cells differentiated into macrophage-like cells when monocytes were supplemented with oxidized LDL alone. CONCLUSIONS: The finding provides preliminary evidence on regulation of cell growth and differentiation in monocyte and monocyte-derived macrophages by Nigella sativa oil. Further investigations need to be conducted to explain its mechanism in human monocyte.
Subject(s)
Cell Differentiation/drug effects , Cell Enlargement/drug effects , Monocytes/physiology , Nigella sativa , Plant Oils/pharmacology , CD11b Antigen/metabolism , Cells, Cultured , Humans , Lipid Metabolism , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/pharmacology , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Primary Cell CultureABSTRACT
Isoflavones are ubiquitous compounds in foods and in the environment in general. Daidzein and genistein, the best known of isoflavones, are structurally similar to 17ß-estradiol and known to exert estrogenic effects. They also evidence a broad variety of biological properties, including antioxidant, anti-carcinogenic, anti-atherogenic and anti-osteoporotic activities. Previously, daidzein was reported to increase the phagocytic activity of peritoneal macrophages and splenocyte proliferation, and to inhibit nitric oxide (NO) production in macrophages. However, its potential impacts on immune response in dendritic cells (DCs), antigen-presenting cells that link innate and adaptive immunity, have yet to be clearly elucidated. In this study, we evaluated the effects of isoflavones on the maturation and activation of DCs. Isoflavones (formononetin, daidzein, equol, biochanin A, genistein) were found to differentially affect the expression of CD86, a costimulatory molecule, on lipopolysaccharide (LPS)-stimulated DCs. In particular, daidzein significantly and dose-dependently inhibited the expression levels of maturation-associated cell surface markers including CD40, costimulatory molecules (CD80, CD86), and major histocompatibility complex class II (I-A(b)) molecule on LPS-stimulated DCs. Daidzein also suppressed pro-inflammatory cytokine production such as IL-12p40, IL-6 and TNF-α, whereas it didn't affect IL-10 and IL-1ß expression. Furthermore, daidzein enhanced endocytosis and inhibited the allo-stimulatory ability of LPS-stimulated DCs on T cells, indicating that daidzein treatment can inhibit the functional maturation of DCs. These results demonstrate that daidzein may exhibit immunosuppressive activity by inhibiting the maturation and activation of DCs.
Subject(s)
Cell Enlargement/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , Growth Inhibitors/pharmacology , Isoflavones/pharmacology , Phytoestrogens/pharmacology , Animals , Dendritic Cells/physiology , Female , Immunosuppressive Agents/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
Increasing evidences implicate impairment of axonal integrity in mechanisms underlying neurodegenerative disorders. Beta-boswellic acid (BBA) is the major component of Boswellia serrata gum. This resin has long been used in Ayurveda (India's traditional medicine) to prevent amnesia. In this study, the effect of BBA was examined on neurites outgrowth and branching as well as on polymerization dynamics of tubulin. The morphometric parameters (axonal length and neuritis branching) were examined microscopically after treating the hippocampal cells with BBA. Also the assembly process of tubulin was assessed using UV/V is spectrophotometer through following of absorbance at 350 nm. The results revealed that BBA could significantly enhance neurite outgrowth, branching, and tubulin polymerization dynamics. The obtained results suggest that enhancing effect of BBA on microtubule polymerization kinetics might be the origin of increasing axonal outgrowth and branching.
Subject(s)
Hippocampus/drug effects , Neurites/drug effects , Neurons/drug effects , Nootropic Agents/pharmacology , Triterpenes/pharmacology , Animals , Axons/drug effects , Axons/physiology , Cell Enlargement/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Hippocampus/cytology , Hippocampus/physiology , Kinetics , Microtubules/metabolism , Neurites/physiology , Neurons/cytology , Neurons/physiology , Nootropic Agents/administration & dosage , Rats , Rats, Sprague-Dawley , Spectrophotometry, Ultraviolet , Time Factors , Triterpenes/administration & dosage , Tubulin/metabolismABSTRACT
OBJECTIVE: Phytoestrogens display an array of pharmacologic properties, and in recent years investigation of their potential as anticancer agents has increased dramatically. In this article we review the published literature related to phytoestrogens and breast cancer as well as suggest the possible mechanisms that may underlie the relationship between phytoestrogens and breast cancer. DATA SOURCES: Electronic searches on phytoestrogens and breast cancer were performed on MEDLINE and EMBASE in June 2007. No date restriction was placed on the electronic search. DATA EXTRACTION: We focused on experimental data from published studies that examined the characteristics of phytoestrogens using in vivo or in vitro models. We also include human intervention studies in this review. DATA SYNTHESIS: We evaluated evidence regarding the possible mechanisms of phytoestrogen action. Discussions of these mechanisms were organized into those activities related to the estrogen receptor, cell growth and proliferation, tumor development, signaling pathways, and estrogen-metabolizing enzymes. CONCLUSIONS: We suggest that despite numerous investigations, the mechanisms of phytoestrogen action in breast cancer have yet to be elucidated. It remains uncertain whether these plant compounds are chemoprotective or whether they may produce adverse outcomes related to breast carcinogenesis.
Subject(s)
Anticarcinogenic Agents/pharmacology , Breast Neoplasms/prevention & control , Phytoestrogens/pharmacology , Animals , Anticarcinogenic Agents/adverse effects , Anticarcinogenic Agents/classification , Aromatase/genetics , Aromatase/metabolism , Breast Neoplasms/chemically induced , Breast Neoplasms/metabolism , Cell Enlargement/drug effects , Cell Proliferation/drug effects , Cytochrome P-450 Enzyme System/metabolism , Diet , Estrogens/biosynthesis , Humans , Phytoestrogens/adverse effects , Phytoestrogens/classification , Receptors, Estrogen/metabolism , Risk Factors , Signal TransductionABSTRACT
Following spinal cord injury, a variety of inhibitory molecules hinder the success of axon regeneration. The motile tip of the axon, the growth cone, shares a similar cytoskeletal array as a migrating cell, and in general the cytoskeleton is regulated by a conserved set of signaling pathways that act downstream of guidance cue and growth factor receptors. We exploit these similarities by using migrating cells as a model system to screen for extracts that promote axon outgrowth. The screen is a high-throughput wound-healing assay performed by a 96-pin tool Biogrid robot where positive candidates are identified as extracts that stimulate complete wound healing. Testing of positive candidates on chick DRG explants has lead to the identification of extracts that promote neurite outgrowth on permissive and inhibitory substrates. Extracts can be fractionated to purity, identifying novel compounds that promote neurite outgrowth on inhibitory substrates.
Subject(s)
Cell Migration Assays/methods , Cell Movement/drug effects , Drug Evaluation, Preclinical/methods , Growth Cones/drug effects , Nerve Growth Factors/isolation & purification , Neurites/drug effects , Animals , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Enlargement/drug effects , Cell Extracts/isolation & purification , Cell Extracts/pharmacology , Cell Migration Assays/instrumentation , Cell Movement/physiology , Cells, Cultured , Chick Embryo , Cytoskeleton/drug effects , Cytoskeleton/physiology , Cytoskeleton/ultrastructure , Drug Evaluation, Preclinical/instrumentation , Growth Cones/physiology , Growth Cones/ultrastructure , Growth Inhibitors/pharmacology , Humans , Nerve Growth Factors/pharmacology , Neurites/physiology , Neurites/ultrastructure , Wound Healing/drug effects , Wound Healing/physiologyABSTRACT
The effects of iron, zinc, copper, manganese, and Methocel on the growth of L cells in a synthetic medium were studied. The medium was iron-deficient unless a supplement of 1.0 g/liter Methocel HG (4,000 centipoises), Methocel ash equivalent to 1.0 g/liter Methocel, or 1.0 microM FeSO4 was added. Cells grew faster in the presence of FeSO4 or Methocel ash (generation time = 40 hours) than of Methocel (generation time = 110 hours), but Methocel was needed to protect cells from mechanical destruction. Treating the growth medium with Dowex A-1 chelating resin produced a medium deficient in both iron and zinc. The iron requirement in this medium varied between 0.6 and 1.4 microM, depending on the concentrations of other cations added to the medium. A copper concentration of at least 0.4 microM was beneficial as it reduced the amount of iron necessary for cell growth. No manganese was required in this medium; any added manganese produced an inhibition of cell growth. Additional iron could reverse this inhibition. The minimum iron requirement for the production of L cells was 3 nmoles/mg cell nitrogen produced. Approximately 0.6 microM zinc was required for maximum cell growth in resin-treated medium.