ABSTRACT
Stem cell-based therapies have demonstrated significant potential in clinical applications for many debilitating diseases. The ability to non-invasively and dynamically track the location and viability of stem cells post administration could provide important information on individual patient response and/or side effects. Multi-modal cell tracking provides complementary information that can offset the limitations of a single imaging modality to yield a more comprehensive picture of cell fate. In this study, mesenchymal stem cells (MSCs) were engineered to express human sodium iodide symporter (NIS), a clinically relevant positron emission tomography (PET) reporter gene, as well as labeled with superparamagnetic iron oxide nanoparticles (SPIOs) to allow for detection with magnetic particle imaging (MPI). MSCs were additionally engineered with a preclinical bioluminescence imaging (BLI) reporter gene for comparison of BLI cell viability data to both MPI and PET data over time. MSCs were implanted into the hind limbs of immunocompromised mice and imaging with MPI, BLI and PET was performed over a 30-day period. MPI showed sensitive detection that steadily declined over the 30-day period, while BLI showed initial decreases followed by later rapid increases in signal. The PET signal of MSCs was significantly higher than the background at later timepoints. Early-phase imaging (day 0-9 post MSC injections) showed correlation between MPI and BLI data (R2 = 0.671), while PET and BLI showed strong correlation for late-phase (day 10-30 post MSC injections) imaging timepoints (R2 = 0.9817). We report the first use of combined MPI and PET for cell tracking and show the complementary benefits of MPI for sensitive detection of MSCs early after implantation and PET for longer-term measurements of cell viability.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Mice , Animals , Humans , Mesenchymal Stem Cell Transplantation/methods , Positron-Emission Tomography/methods , Genes, Reporter , Magnetic PhenomenaABSTRACT
This study aimed to examine if methanolic extracts of Pulsatilla vulgaris Mill. can inhibit HeLa cell proliferation through the modulation of cancer-related signaling pathways. The cytotoxicity and chemical composition of P. vulgaris leaves and root extracts were also determined. Research showed that root extract of P. vulgaris inhibited 12 signaling pathways in a cervical cancer cell line and the most potent activation inhibition was observed for MYC, Notch, Wnt, E2F, Ets, Stat3, Smad, Hdghog, AP-1, and NF-κB, at a concentration of 40 µg/mL. The methanolic extracts of P. vulgaris enhanced apoptotic death and deregulated cellular proliferation, differentiation, and progression toward the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. This is the first study to report the influence of P. vulgaris on cancer signaling pathways. Additionally, our detailed phytochemical analysis of the methanolic extracts of P. vulgaris gives a conclusion that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells were mainly triterpenoid saponins accompanied by phenolic acids.
Subject(s)
Neoplasms , Pulsatilla , Humans , HeLa Cells , Genes, Reporter , Signal Transduction , Cell Proliferation , Plant Extracts/pharmacology , Plant Extracts/chemistry , Cell Line, Tumor , ApoptosisABSTRACT
Four new embryonic cell lines derived from blastocysts of the olive flounder Paralichthys olivaceus, an important commercial marine fish, were established and characterized. They were designated as PoEFCI, PoEFCII, PoEFCIII, and PoEFCIV and were all fibroblastic cells. The cells were cultured in DMEM/F-12 medium supplemented with antibiotics, FBS, and growth factors at temperature of 25 °C and subcultured for >100 passages over 18 months. The origin of the cell lines was confirmed by examining the partial sequences of the cytochrome oxidase c subunit I (COI) gene of the flounder mitochondrial DNA (mtDNA). The four cell lines showed different growth curve patterns. According to the results of gene and protein expression and enzyme activity, the cell lines PoEFCI, PoEFCII, and PoEFC III could be pluripotent. The cells of all four cell lines were also successfully transfected with the green fluorescent protein (GFP) reporter gene, suggesting that they could be used to study gene function in the flounder or other fish. More importantly, PoEFCI-III were sensitive to chromium (Cr) and red sea bream Pagrus major iridovirus (RSIV), so they could be used as a powerful tool for the study of the toxicological investigation of heavy metals and RSIV in fish. Therefore, these cell lines would be useful for biotechnological and toxicological research on marine fish as an in vitro biological system.
Subject(s)
Fish Diseases , Flounder , Animals , Flounder/genetics , Cell Line , Green Fluorescent Proteins/genetics , Genes, Reporter , Fish Diseases/geneticsABSTRACT
Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).
Subject(s)
Codon, Nonsense , Genetic Diseases, Inborn , Guanidines , Quinazolines , Cell Line , Codon, Nonsense/drug effects , Codon, Nonsense/genetics , Codon, Terminator/drug effects , Codon, Terminator/genetics , Drug Evaluation, Preclinical , Genes, Reporter/drug effects , Genetic Diseases, Inborn/drug therapy , Genetic Diseases, Inborn/genetics , Gentamicins/pharmacology , Guanidines/pharmacology , High-Throughput Screening Assays , Humans , Muscular Dystrophy, Duchenne/drug therapy , Muscular Dystrophy, Duchenne/genetics , Quinazolines/pharmacologyABSTRACT
The mitogen-activated protein kinase OsMPK1 is involved in abscisic acid (ABA) biosynthesis in rice (Oryza sativa L.). However, the underlying molecular mechanisms of OsMPK1 in regulating ABA biosynthesis are poorly understood. Here, by using yeast two-hybrid assay and firefly luciferase complementary imaging assay, we show that OsMPK1 physically interact with a short-chain dehydrogenase protein OsABA2. However, OsMPK5, a homolog of OsMPK1, does not interact with OsABA2. Further, OsMPK1 can phosphorylate OsABA2S197 in vitro. Phosphorylation at the position of OsABA2S197 does not affect its subcellular localization, but enhances the stability of OsABA2 protein. We also found that OsABA2 has feedback regulation on OsMPK1 kinase activity. Further research reveals that OsMPK1 and OsABA2 coordinately regulate the biosynthesis of ABA, and phosphorylation of OsABA2 at Ser197 by OsMPK1 plays a crucial role in regulating the biosynthesis of ABA. Finally, genetic analysis showed that OsABA2 can enhance the sensitivity of rice to ABA and the tolerance of rice to drought and salt stress.
Subject(s)
Abscisic Acid/metabolism , Alcohol Oxidoreductases/genetics , Mitogen-Activated Protein Kinases/genetics , Oryza/genetics , Plant Proteins/genetics , Recombinant Proteins/genetics , Alcohol Oxidoreductases/metabolism , Droughts , Feedback, Physiological , Gene Expression Regulation, Plant , Genes, Reporter , Isoenzymes/genetics , Isoenzymes/metabolism , Luciferases/genetics , Luciferases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Onions/genetics , Onions/metabolism , Oryza/metabolism , Phosphorylation , Plant Growth Regulators/metabolism , Plant Proteins/metabolism , Plants, Genetically Modified , Protein Processing, Post-Translational , Protein Stability , Recombinant Proteins/metabolism , Signal Transduction , Stress, Physiological , Two-Hybrid System TechniquesABSTRACT
Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.
Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Directed Molecular Evolution/methods , Escherichia coli/genetics , Phenylalanine/metabolism , Protein Biosynthesis , RNA, Transfer/genetics , Amino Acids/metabolism , Amino Acids/pharmacology , Amino Acyl-tRNA Synthetases/metabolism , Animals , Base Pairing , Biomimetic Materials/metabolism , Biomimetic Materials/pharmacology , Cell Engineering , Escherichia coli/metabolism , Gene Expression , Genes, Reporter , Germ-Free Life , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Nucleic Acid Conformation , Phenylalanine/pharmacology , Plasmids/chemistry , Plasmids/metabolism , RNA, Transfer/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Adenovirus (Ad) has risen to be a promising alternative to conventional cancer therapy. However, systemic delivery of Ad, which is necessary for the treatment of metastatic cancer, remains a major challenge within the field, owing to poor tumor tropism and nonspecific hepatic tropism of the virus. To address this limitation of Ad, we have synthesized two variants of folic acid (FA)-conjugated methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-L-glutamate (P5N2LG-FA and P5N5LG-FA) using 5 kDa poly(ethylene glycol) (PEG) with a different level of protonation (N2 < N5 in terms of charge), along with a P5N5LG control polymer without FA. Our findings demonstrate that P5N5LG, P5N2LG-FA, and P5N5LG-FA exert a lower level of cytotoxicity compared to 25 kDa polyethyleneimine. Furthermore, green fluorescent protein (GFP)-expressing Ad complexed with P5N2LG-FA and P5N5LG-FA (Ad/P5N2LG-FA and Ad/P5N5LG-FA, respectively) exerted superior transduction efficiency compared to naked Ad or Ad complexed with P5N5LG (Ad/P5N5LG) in folate receptor (FR)-overexpressing cancer cells (KB and MCF7). All three nanocomplexes (Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA) internalized into cancer cells through coxsackie adenovirus receptor-independent endocytic mechanism and the cell uptake was more efficient than naked Ad. Importantly, the cell uptake of the two FA functionalized nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) was dependent on the complementary interaction of FA-FR. Systemically administered Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA showed exponentially higher retainment of the virus in blood circulation up to 24 h post-administration compared with naked Ad. Both tumor-targeted nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) showed significantly higher intratumoral accumulation than naked Ad or Ad/P5N5LG via systemic administration. Both tumor-targeted nanocomplexes accumulated at a lower level in liver tissues compared to naked Ad. Notably, the nonspecific accumulation of Ad/P5N2LG-FA was significantly lower than Ad/P5N5LG-FA in several normal organs, while exhibiting a significantly higher intratumoral accumulation level, showing that careful optimization of polyplex surface charge is critical to successful tumor-targeted systemic delivery of Ad nanocomplexes.
Subject(s)
Adenoviridae/genetics , Biocompatible Materials/chemistry , Genetic Vectors , Nanoparticles , Neoplasms/genetics , Polymers/chemistry , Transduction, Genetic , A549 Cells , Adenoviridae/metabolism , Animals , Gene Expression Regulation, Neoplastic , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , MCF-7 Cells , Male , Mice, Nude , Neoplasms/metabolism , Surface Properties , Tissue DistributionABSTRACT
Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (valproate) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with valproate improved the swimming of the MJD zebrafish, affected levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that valproate treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in valproate treated MJD zebrafish and HEK293 cells expressing ataxin-3 84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with valproate alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by valproate and the beneficial effects of valproate on the movement in the MJD zebrafish, supporting that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.
Subject(s)
Histone Deacetylase Inhibitors/therapeutic use , Machado-Joseph Disease/drug therapy , Sirtuins/drug effects , Valproic Acid/therapeutic use , Acetylation , Animals , Animals, Genetically Modified , Ataxin-3/antagonists & inhibitors , Ataxin-3/genetics , Ataxin-3/metabolism , Autophagy/drug effects , Carbazoles/pharmacology , Carbazoles/therapeutic use , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Synergism , Genes, Reporter , HEK293 Cells , Histone Deacetylase Inhibitors/pharmacology , Histones/metabolism , Humans , Peptides/genetics , Protein Processing, Post-Translational , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Resveratrol/pharmacology , Resveratrol/therapeutic use , Signal Transduction , Sirtuin 1/physiology , Sirtuins/physiology , Swimming , Trinucleotide Repeat Expansion , Valproic Acid/pharmacology , Zebrafish , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Inhibition of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome has recently emerged as a promising therapeutic target for several inflammatory diseases. After priming and activation by inflammation triggers, NLRP3 forms a complex with apoptosis-associated speck-like protein containing a CARD domain (ASC) followed by formation of the active inflammasome. Identification of inhibitors of NLRP3 activation requires a well-validated primary high-throughput assay followed by the deployment of a screening cascade of assays enabling studies of structure-activity relationship, compound selectivity and efficacy in disease models. We optimized a NLRP3-dependent fluorescent tagged ASC speck formation assay in murine immortalized bone marrow-derived macrophages and utilized it to screen a compound library of 81,000 small molecules. Our high-content screening assay yielded robust assay metrics and identified a number of inhibitors of NLRP3-dependent ASC speck formation, including compounds targeting HSP90, JAK and IKK-ß. Additional assays to investigate inflammasome priming or activation, NLRP3 downstream effectors such as caspase-1, IL-1ß and pyroptosis form the basis of a screening cascade to identify NLRP3 inflammasome inhibitors in drug discovery programs.
Subject(s)
Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Inflammasomes/drug effects , Macrophages/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Animals , CARD Signaling Adaptor Proteins/metabolism , Caspase 1/biosynthesis , Cells, Cultured , Dimethyl Sulfoxide/pharmacology , Drug Discovery , Furans/pharmacology , Genes, Reporter , Indenes/pharmacology , Interleukin-1beta/biosynthesis , Lipopolysaccharides/pharmacology , Mice , Nigericin/pharmacology , Phenotype , Pyroptosis/drug effects , Recombinant Proteins/metabolism , Small Molecule Libraries , Sulfonamides/pharmacologyABSTRACT
Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the "common" TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose-response, by limited structure-activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.
Subject(s)
Drug Discovery/methods , Enzyme Stability/drug effects , High-Throughput Screening Assays/methods , Small Molecule Libraries , Triose-Phosphate Isomerase/chemistry , Drug Evaluation, Preclinical/methods , Genes, Reporter , HEK293 Cells , Humans , Mutation , Triose-Phosphate Isomerase/antagonists & inhibitors , Triose-Phosphate Isomerase/deficiency , Triose-Phosphate Isomerase/geneticsABSTRACT
Triterpenes (30-carbon isoprene compounds) represent a large and highly diverse class of natural products that play various physiological functions in plants. The triterpene biosynthetic enzymes, particularly those catalyzing the late-stage regio-selective modifications are not well characterized. The bark of select Boswellia trees, e.g., B. serrata exudes specialized oleo-gum resin in response to wounding, which is enriched with boswellic acids (BAs), a unique class of C3α-epimeric pentacyclic triterpenes with medicinal properties. The bark possesses a network of resin secretory structures comprised of vertical and horizontal resin canals, and amount of BAs in bark increases considerably in response to wounding. To investigate BA biosynthetic enzymes, we conducted tissue-specific transcriptome profiling and identified a wound-responsive BAHD acetyltransferase (BsAT1) of B. serrata catalyzing the late-stage C3α-O-acetylation reactions in the BA biosynthetic pathway. BsAT1 catalyzed C3α-O-acetylation of αBA, ßBA, and 11-keto-ßBA in vitro and in planta assays to produce all the major C3α-O-acetyl-BAs (3-acetyl-αBA, 3-acetyl-ßBA, and 3-acetyl-11-keto-ßBA) found in B. serrata bark and oleo-gum resin. BsAT1 showed strict specificity for BA scaffold, whereas it did not acetylate the more common C3ß-epimeric pentacyclic triterpenes. The analysis of steady-state kinetics using various BAs revealed distinct substrate affinity and catalytic efficiency. BsAT1 transcript expression coincides with increased levels of C3α-O-acetyl-BAs in bark in response to wounding, suggesting a role of BsAT1 in wound-induced biosynthesis of C3α-O-acetyl-BAs. Overall, the results provide new insights into the biosynthesis of principal chemical constituents of Boswellia oleo-gum resin.
Subject(s)
Acetyltransferases/metabolism , Boswellia/enzymology , Resins, Plant/metabolism , Transcriptome , Triterpenes/metabolism , Acetyltransferases/genetics , Biosynthetic Pathways , Boswellia/anatomy & histology , Boswellia/chemistry , Boswellia/genetics , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Genes, Reporter , Organ Specificity , Plant Bark/anatomy & histology , Plant Bark/chemistry , Plant Bark/enzymology , Plant Bark/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Medicinal , Resins, Plant/chemistry , Nicotiana/genetics , Nicotiana/metabolism , Triterpenes/chemistryABSTRACT
Demethoxycurcumin (DMC), a derivate of curcumin, has been shown to induce apoptotic cell death in human glioblastoma multiforme GBM 8401 cells via cell cycle arrest and induction of cell apoptosis. However, there is no report showing DMC suppresses glioblastoma multiforme cells in vivo. In the present study, we investigated the effects of DMC on GBM8401 cells in vivo. At first, we established a luciferase-expressing stable clone named GBM 8401/luc2. Second, mice were inoculated subcutaneously with GBM 8401/luc2 cells to generate a xenograft tumor mice model. After inoculation, tumor volume reached 100-120 mm3, and all mice were randomly divided into three groups: Group I was treated with 110 µL phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 30 mg/kg of DMC, and Group III with 60 mg/kg of DMC. Mice from each group were given the oral treatment of DMC by gavage for 21 days. The body weight and tumor volume were recorded every 3 days. DMC significantly decreased the tumor volumes, and 60 mg/kg treatment showed a higher decrease in tumor volumes than that of 30 mg/kg, However, DMC did not affect the body weights. The photons emitted from mice tumors were detected with Xenogen IVIS imaging system, DMC at both doses decreased the total photon flux and 60 mg/kg treatment of DMC has low total photon flux than that of 30 mg/kg. The tumor volumes and weights in 60 mg/kg treatment of DMC were lower than that of 30 mg/kg. Immunohistochemical analysis was used to measure protein expression of tumors and results showed that DMC treatment led to lightly staining with anti-Bcl-2 and -XIAP and 60 mg/kg treatment of DMC has lighter staining with anti-Bcl-2 and -XIAP than that of 30 mg/kg. The higher dose (60 mg/kg) of DMC has higher signals of cleaved-caspase-3 than that of the lower dose (30 mg/kg). Furthermore, the hematoxylin and eosin (H&E) staining of liver tissues showed no significant difference between DMC-treated and control-groups. Overall, these observations showed that DMC suppressed tumor properties in vivo and DMC may be used against human glioblastoma multiforme in the future.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Brain Neoplasms/drug therapy , Diarylheptanoids/therapeutic use , Glioblastoma/drug therapy , Animals , Antineoplastic Agents, Phytogenic/toxicity , Apoptosis/drug effects , Brain Neoplasms/metabolism , Cell Line, Tumor , Diarylheptanoids/toxicity , Genes, Reporter , Glioblastoma/metabolism , Humans , Liver/drug effects , Liver/pathology , Male , Mice , Mice, Nude , Neoplasm Proteins/analysis , Proto-Oncogene Proteins c-bcl-2/analysis , Random Allocation , Tumor Burden , X-Linked Inhibitor of Apoptosis Protein/analysis , Xenograft Model Antitumor Assays , bcl-2-Associated X Protein/analysisABSTRACT
More than 30% of all pharmaceuticals target G-protein-coupled receptors (GPCRs). Here, we present a GPCR-based screen in yeast to identify ligands for human serotonin receptor 4 (5-HTR4). Serotonin receptor 4 agonists are used for the treatment of irritable bowel syndrome with constipation. Specifically, the HTR4-based screen couples activation of 5-HTR4 on the yeast cell surface to luciferase reporter expression. The HTR4-based screen has a throughput of one compound per second allowing the screening of more than a thousand compounds per day.
Subject(s)
Receptors, G-Protein-Coupled/agonists , Receptors, Serotonin, 5-HT4/chemistry , Saccharomyces cerevisiae/metabolism , Serotonin 5-HT4 Receptor Agonists/pharmacology , Drug Evaluation, Preclinical , Genes, Reporter , HEK293 Cells , High-Throughput Screening Assays/methods , Humans , Ligands , Luciferases/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Serotonin, 5-HT4/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
Reporter gene assays are powerful tools for monitoring dynamic molecular changes and for evaluating the responses that occur at the genetic elements within cells in response to exogenous molecules. In general, various protein systems can be used as reporter genes, including luciferases. Here, the present protocol introduces a unique reporter gene system for monitoring molecular events in cells using bacterial luciferase (lux), which can generate blue-green light suitable for gene reporter applications with the highest cost performance. The protocol also guides the assay conditions and necessary components for using of lux gene (lux) as a eukaryotic reporter system. The lux system can be applied to monitor variety of molecular events inside mammalian cellular systems.
Subject(s)
Drug Evaluation, Preclinical/methods , Genes, Reporter , Luciferases, Bacterial/metabolism , Luminescent Measurements/methods , Genetic Vectors , HEK293 Cells , Humans , Luciferases, Bacterial/drug effects , Luciferases, Bacterial/geneticsABSTRACT
Estradiol (E2) and selective estrogen receptor modulators (SERMs) have broad-ranging cellular effects that include mitochondrial respiration and reactive oxygen species (ROS) metabolism. Many of these effects have been studied using cell culture models. Recent advances have revealed the extent to which cellular metabolism is affected by the culture environment. Cell culture media with metabolite composition similar to blood plasma [e.g., Plasmax, Human Plasma-Like Medium (HPLM)] alter cellular behaviors including responses to drugs. Similar effects have been observed with respect to O2 levels in cell culture. Given these observations, we investigated whether the effects of E2 and SERMs are also influenced by media composition and O2 level during cell culture experiments. We analyzed mitochondrial network characteristics, cellular oxidative metabolism, and H2O2 production in C2C12 myoblasts growing in physiological (5%) or standard cell culture (18%)O2 and in physiological (Plasmax) or standard cell culture [Dulbecco's modified Eagle's medium (DMEM)] media. Although E2 significantly lowered H2O2 production from cells growing in 18% O2/DMEM (standard cell culture), it had no effect on cells growing in Plasmax. Moreover, culture conditions significantly altered the effects of E2 and SERMs on mitochondrial abundance and network characteristics. These results indicate that the effects of E2 and SERMs on various aspects of cell physiology strongly depends on growth conditions, which in turn emphasizes the need to consider this carefully in cell culture experiments.
Subject(s)
Culture Media/pharmacology , Estradiol/pharmacology , Mitochondria/drug effects , Myoblasts/drug effects , Oxygen/pharmacology , Reactive Oxygen Species/metabolism , Animals , Apoptosis , Cell Culture Techniques , Cell Line , Culture Media/chemistry , Fluorescence , Genes, Reporter , Hydrogen Peroxide/metabolism , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolismABSTRACT
Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Carcinoma, Hepatocellular/pathology , Gene Expression Regulation, Neoplastic/drug effects , Lactones/pharmacology , Liver Neoplasms/pathology , Neoplasm Proteins/biosynthesis , STAT3 Transcription Factor/biosynthesis , Sesquiterpenes/pharmacology , Transcriptional Activation/drug effects , Cell Line, Tumor , Down-Regulation/drug effects , Drug Screening Assays, Antitumor , Genes, Reporter , Humans , Neoplasm Proteins/genetics , Oxidative Stress , Protein Kinase Inhibitors/pharmacology , Recombinant Proteins/metabolism , STAT3 Transcription Factor/genetics , Sorafenib/pharmacologyABSTRACT
The purpose of this study was to determine if a methanolic extract of the Pulsatilla patens (L.) Mill. can inhibit the progression of cancer through the modulation of cancer-related metabolic signaling pathways. We analyzed a panel of 13 inducible luciferase reporter gene vectors which expression is driven by enhancer elements that bind to specific transcription factors for the evaluation of the activity of cancer signaling pathways. The root extract of P. patens exhibited strong inhibition of several signaling pathways in HeLa cells, a cervical cancer cell line, and was found to be the most potent in inhibiting the activation of Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt and Hdghog, at a concentration of 40 µg/mL. The methanolic extracts of P. patens enhanced apoptotic death, deregulated cellular proliferation, differentiation, and progression towards the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. This is the first study to report the influence of Pulsatilla species on cancer signaling pathways. Further, our detailed phytochemical analysis of the methanolic extracts of the P. patens allowed to deduce that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells were mainly triterpenoid saponins accompanied by phenolic acids.
Subject(s)
Neoplasms/metabolism , Plant Extracts/pharmacology , Pulsatilla/chemistry , Signal Transduction , Cell Death/drug effects , Coumaric Acids/pharmacology , Genes, Reporter , HeLa Cells , Humans , Limit of Detection , Luciferases/metabolism , Methanol , Neoplasm Proteins/metabolism , Neoplasms/pathology , Plant Roots/chemistry , Reproducibility of Results , Saponins/chemistry , Saponins/pharmacology , Signal Transduction/drug effects , Triterpenes/chemistry , Triterpenes/pharmacologyABSTRACT
In a disease-state-dependent manner, the histamine-resistant itch in dry skin-based skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Our previous studies have shown that Sema3A ointment inhibits epidermal hyperinnervation and scratching behavior and improves dermatitis scores in AD model mice. Therefore, we consider Sema3A as a key therapeutic target for improving histamine-resistant itch in AD and xerosis. This study was designed to screen a library of herbal plant extracts to discover compounds with potential to induce Sema3A in normal human epidermal keratinocytes (NHEKs) using a reporter gene assay, so that positive samples were found. Among the positive samples, only the extract of S. baicalensis was found to consistently increase Sema3A levels in cultured NHEKs in assays using quantitative real-time PCR and ELISA. In evaluation of reconstituted human epidermis models, the level of Sema3A protein in culture supernatants significantly increased by application of the extract of S. baicalensis. In addition, we investigated which components in the extract of S. baicalensis contributed to Sema3A induction and found that baicalin and baicalein markedly increased the relative luciferase activity, and that baicalein had higher induction activity than baicalin. Thus, these findings suggest that S. baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes.
Subject(s)
Plant Extracts/chemistry , Scutellaria baicalensis/chemistry , Semaphorin-3A/metabolism , Cell Line , Flavanones/genetics , Flavanones/metabolism , Flavonoids/genetics , Flavonoids/metabolism , Genes, Reporter , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Models, Biological , Plant Extracts/pharmacology , RNA, Messenger/metabolism , Scutellaria baicalensis/metabolism , Semaphorin-3A/geneticsABSTRACT
Biophysical methods are widely employed in academia and the pharmaceutical industry to detect and quantify weak molecular interactions. Such methods find broad application in fragment-based drug discovery (FBDD). In an FBDD campaign, a suitable affinity determination method is key to advancing a project beyond the initial screening phase. Protein-observed (PO) nuclear magnetic resonance (NMR) finds widespread use due to its ability to sensitively detect very weak interactions at residue-level resolution. When there are issues precluding the use of PO-NMR, ligand-observed (LO) NMR reporter assays can be a useful alternative. Such assays can measure affinities in a similar range to PO-NMR while offering some distinct advantages, especially with regard to protein consumption and compound throughput. In this paper, we take a closer look at setting up such assays for routine use, with the aim of getting high-quality, accurate data and good throughput. We assess some of the key characteristics of these assays in the mathematical framework established for fluorescence polarization assays with which the readers may be more familiar. We also provide guidance on setting up such assays and compare their performance with other affinity determination methods that are commonly used in drug discovery.
Subject(s)
Drug Discovery/methods , Genes, Reporter , Ligands , Magnetic Resonance Spectroscopy/methods , Proteins/chemistry , Biological Assay , Drug Evaluation, Preclinical , Fluorescence Polarization/methods , Humans , Protein Binding , Proteins/metabolismABSTRACT
A yeast estrogen screening (YES) assay was improved to increase sensitivity for detection of phytoestrogens. New yeast strains minus one or the other of transporters Pdr5 or Snq2 and harboring yEGFP as a reporter gene were developed. The new strains showed 2-100-fold improvement in sensitivity for detection of standard estrogens and antiestrogens. In addition, the assay time (1 h) using the newly developed strains was shorter than that (4 h) previously reported. Furthermore, the snq2-minus strains were most effective for detection of estrogenic activity while the pdr5-minus strains were most effective for detection of antiestrogenic activity. The efficacy of the new methods was evaluated and confirmed by testing with 23 Thai medicinal plant species. The new strains were also tested for detection of xenoestrogens. The results revealed that the newly developed YES methods were specific and rapid and suitable for simple high-throughput screening or detection of estrogen-like compounds.