ABSTRACT
MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/ß-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.
Subject(s)
Activation, Metabolic/drug effects , DNA Damage/drug effects , Mutagenicity Tests/methods , Mutagens/toxicity , Animals , Anisomycin/toxicity , Brefeldin A/toxicity , Cell Line , Cycloheximide/toxicity , High-Throughput Screening Assays/methods , Histones/genetics , Humans , Liver/drug effects , Liver/metabolism , Rats , Tumor Suppressor Protein p53/geneticsABSTRACT
Endoplasmic reticulum (ER) stress and major chemotherapeutic agents damage DNA by generating reactive oxygen species (ROS). Here we show that ER stress and chemotherapy induce leukotriene C4 (LTC4) biosynthesis by transcriptionally upregulating and activating the enzyme microsomal glutathione-S-transferase 2 (MGST2) in cells of non-haematopoietic lineage. ER stress and chemotherapy also trigger nuclear translocation of the two LTC4 receptors. Acting in an intracrine manner, LTC4 then elicits nuclear translocation of NADPH oxidase 4 (NOX4), ROS accumulation and oxidative DNA damage. Mgst2 deficiency, RNAi and LTC4 receptor antagonists abolish ER stress- and chemotherapy-induced ROS and oxidative DNA damage in vitro and in mouse kidneys. Cell death and mouse morbidity are also significantly attenuated. Hence, MGST2-generated LTC4 is a major mediator of ER stress- and chemotherapy-triggered oxidative stress and oxidative DNA damage. LTC4 inhibitors, commonly used for asthma, could find broad clinical use in major human pathologies associated with ER stress-activated NOX4.
Subject(s)
DNA Damage , Leukotriene C4/metabolism , Oxidative Stress , Animals , Brefeldin A/toxicity , Cell Survival , Cells, Cultured , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/physiology , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Kidney Diseases/chemically induced , Leukotriene C4/genetics , Mice , Mice, Knockout , RNA Interference , Tunicamycin/toxicityABSTRACT
Brefeldin A has attracted considerable attention because of its potential function in cancer prevention. However, its therapeutic use is limited by its poor bioavailability. The modifications on brefeldin A were difficult because of its low stability and selectivity toward two hydroxyl groups within the same molecule. In this study, we report the selective acylation of brefeldin A under mild conditions and the preparation of a series of monoacylated and diacylated brefeldin A derivatives. Their cytotoxicity, antitumor activity against TE-1 cell, and molecular properties of adsorption, distribution, metabolism, and elimination were evaluated. Brefeldin A 7-O-benzoate, brefeldin A 4,7-O-dibenzoate, and brefeldin A 7-O-biotin carboxylate showed the most potent cytotoxic activity, with GI50 values of 0.39, 0.46, and 0.50 µm, respectively. Molecular docking of these analogs revealed that the derivatives were well tolerated at the interface between ARF1 and its guanine nucleotide exchange factor ARNO. Our results may serve as a basis for the development of novel potential anticancer agents from brefeldin A derivatives.
Subject(s)
Antineoplastic Agents/chemical synthesis , Brefeldin A/chemistry , ADP-Ribosylation Factor 1/chemistry , ADP-Ribosylation Factor 1/metabolism , Acylation , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/toxicity , Binding Sites , Brefeldin A/blood , Brefeldin A/toxicity , Cell Line, Tumor , Cell Survival/drug effects , Half-Life , Humans , Molecular Docking Simulation , Protein Structure, Tertiary , Rats , Rats, Wistar , Structure-Activity RelationshipABSTRACT
Oryzalin is a much-used pre-emergence herbicide which causes microtubules (Mt) to depolymerize. Here, we document that this dinitroaniline herbicide also leads to characteristic changes in the morphology of the endoplasmic reticulum (ER) and Golgi apparatus. These effects, which are reversible upon washing out the herbicide, are already elicited at low concentrations (2 microM) and become most pronounced at 20 microM. For our studies, we have employed roots of Arabidopsis thaliana, tobacco leaf epidermal cells, and BY-2 suspension cultures, all expressing the luminal ER marker GFP::HDEL. In all cell types, the typical cortical network of the ER assumed a pronounced nodulated morphology with increasing oryzalin concentrations. This effect was enhanced through subsequent application of brefeldin A (BFA). Thin sections of Arabidopsis roots observed in the electron microscope revealed the nodules to consist of a mass of anastomosing ER tubules. Oryzalin also caused the cisternae in Golgi stacks to increase in number but reduced their diameter. Oryzalin retarded ER mobility but did not prevent latrunculin B-induced clustering of Golgi stacks on islands of cisternal ER. While the mechanism underlying these changes in endomembranes remains unknown, it is specific for oryzalin since these effects were not elicited with other Mt-depolymerizing herbicides, e.g., trifluralin, amiprophosmethyl, or colchicine.
Subject(s)
Arabidopsis/drug effects , Dinitrobenzenes/toxicity , Endoplasmic Reticulum/drug effects , Herbicides/toxicity , Microtubules/metabolism , Nicotiana/drug effects , Sulfanilamides/toxicity , Arabidopsis/metabolism , Brefeldin A/toxicity , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Microscopy, Confocal , Microtubules/drug effects , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Roots/drug effects , Plant Roots/metabolism , Nicotiana/metabolismABSTRACT
In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.
Subject(s)
Brefeldin A/metabolism , Cell Death , Filipin/metabolism , Shiga Toxin 1/metabolism , Trihexosylceramides/metabolism , Animals , Brefeldin A/toxicity , Caveolin 1 , Caveolins/metabolism , Cell Survival , Chlorocebus aethiops , Cytoplasm/metabolism , Filipin/toxicity , Golgi Apparatus/metabolism , Protein Biosynthesis , Protein Transport , Shiga Toxin 1/toxicity , Vero CellsABSTRACT
To date, no therapy has been found to which pancreatic cancer responds with the exception of surgical resection in early stages. Recently, gemcitabine has become the standard of care for chemotherapy in those patients with advanced disease. Most pancreatic tumors however, develop resistance to gemcitabine. The aim of this study is to clarify the mechanism of resistance to gemcitabine in human pancreatic cells. Using a cell selection method, a human pancreatic cancer cell line resistant to gemcitabine was established. Cellular proliferation and viability were determined by MTT assay. The cell line with acquired resistance was also found to have cross resistance to fluorouracil. Brefeldin-A (BFA) has been used as a tool for studies of intracellular protein traffic, rather than as an anticancer drug. BFA displays the same effects on wild type cells and those with acquired resistance. Gemcitabine combined with BFA in low doses is significantly more effective than gemcitabine alone against MIA PaCa-2 cell line. Our data suggest that the gemcitabine-resistant and 5-FU-resistant pathways may partially overlap each other. In short, BFA may be used as a modulator of gemcitabine.
Subject(s)
Brefeldin A/pharmacology , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Resistance, Neoplasm , Pancreatic Neoplasms/pathology , Brefeldin A/toxicity , Cell Division/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Drug Interactions , Fluorouracil/pharmacology , Humans , GemcitabineABSTRACT
Our previous studies have shown that human mesangial cells (HMCs) incubated with fibrogenic glomerulopathic monoclonal light chains (G-LCs) obtained from the urine of patients with light chain deposition disease produce increased extracellular matrix (ECM) when compared with HMCs not exposed to fibrogenic LCs. This overproduction of ECM proteins is regulated by transforming growth factor-beta (TGF-beta); blocking TGF-beta normalizes the production of ECM proteins. All ECM proteins, after synthesis, have to go through the secretory pathway in the endoplasmic reticulum (ER) and Golgi complex for final maturation and secretion. Blocking the secretory pathway may reduce the accumulation of ECM proteins. We tested the effect of tunicamycin, a specific inhibitor of N-linked glycosylation in the ER which inhibited glycosylation and brefeldin A, an inhibitor of vesicle transport between the endoplasmic reticulum and the Golgi complex, on ECM protein production, both resulting in subsequent upregulation of glucose-regulated protein 78. Overproduction of fibronectin and tenascin by HMCs was normalized by tunicamycin and brefeldin A. Similarly, when HMCs were exposed to exogenous TGF-beta, the increase in fibronectin was reversed by tunicamycin and brefeldin A. Exogenous platelet-derived growth factor-beta (PDGF-beta) did not induce fibronectin overproduction but significantly stimulated proliferation of HMCs. In summary, this study further supports the notion that fibrogenic G-LCs promote the accumulation of ECM proteins, through the actions of TGF-beta. Importantly, the data indicate that altering protein trafficking in the ER results in impairment of secretion of proteins into the ECM. Furthermore, the data also reveal that PDGF-beta and TGF-beta act independently and that PDGF-beta activation by itself cannot increase ECM proteins directly, but only by increasing the number of HMCs.
Subject(s)
Fibronectins/biosynthesis , Glomerular Mesangium/immunology , Glomerular Mesangium/pathology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/urine , Brefeldin A/pharmacology , Brefeldin A/toxicity , Cell Division/drug effects , Cells, Cultured , Glomerular Mesangium/chemistry , Glomerular Mesangium/drug effects , Humans , Immunoglobulin Light Chains/pharmacology , Immunoglobulin Light Chains/urine , Kidney Glomerulus/immunology , Kidney Tubules/immunology , Proto-Oncogene Proteins c-sis/biosynthesis , Proto-Oncogene Proteins c-sis/pharmacology , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/pharmacology , Tunicamycin/pharmacology , Tunicamycin/toxicityABSTRACT
Brefeldin A (BFA) is a fungus metabolite that is known to cause the disassembly of the Golgi complex and apoptosis in exposed cells, both of which have been suggested as playing roles in the pathogenesis of neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS). This study showed that BFA caused neurotoxicity and apoptotic nuclear changes in cultured spinal neurons of rat spinal cord in a dose- and time-dependent manner. The spinal motor neurons were more vulnerable to this neurotoxicity. The cultured spinal neurons showed irreversible disassembly of the Golgi apparatus as early as 1 hr after exposure to BFA. BFA induced the expression and activation of caspase-12 beginning 8 hr after exposure. The level of the cleaved form of caspase-3 had increased 12 hr after the addition of BFA. Free radical generation and loss of mitochondrial membrane potential were observed in the later stages of neurotoxicity caused by BFA. Collectively, our data suggests that BFA is an excellent agent for reproducing the pathophysiological features of ALS. This in vitro model may be useful in attempts to study the mechanisms of this neurodegenerative disease and to examine therapeutic potentials.
Subject(s)
Brefeldin A/toxicity , Neurons/drug effects , Spinal Cord/drug effects , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Neurons/metabolism , Neurons/pathology , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism , Spinal Cord/pathologyABSTRACT
Paecilomyces sp. and Aspergillus clavatus, which were isolated from Taxus mairei and Torreya grandis from southeast China, produced toxic metabolites when grown in liquid culture. Nuclear magnetic resonance techniques, infrared spectrometry, electrospray ionization mass spectroscopy and X-ray analysis identified brefeldin A, a bioactive metabolite produced by a number of fungal species belonging to the genera Alternaria, Ascochyta, Penicillium, Curvularia, Cercospora and Phyllosticta. This is the first report of the isolation of the cytotoxin from Paecilomyces sp. and A. clavatus. The relevance of brefeldin A to the association between these fungi and their host plants is discussed.
Subject(s)
Aspergillus/metabolism , Brefeldin A/metabolism , Cytotoxins/biosynthesis , Mycotoxins/biosynthesis , Paecilomyces/metabolism , Aspergillus/isolation & purification , Brefeldin A/chemistry , Brefeldin A/toxicity , Cell Line , China , Cytotoxins/chemistry , Cytotoxins/toxicity , Ecosystem , Humans , Microscopy, Electron, Scanning , Molecular Structure , Mycotoxins/chemistry , Mycotoxins/toxicity , Paecilomyces/isolation & purification , Plants, Medicinal/microbiology , Taxus/microbiology , Tracheophyta/microbiologyABSTRACT
Flow cytometry is increasingly used for cytokine detection where it serves to complement ELISA (enzyme-linked immunosorbent assay) and ELISPOT assays. Since it is possible to stain both extracellular epitopes and intracellular cytokines on the same cells, this is a powerful technique for analysing cytokine expression in defined cell populations. However unstimulated cells do not express cytokines. Thus, appropriate stimulation is a prerequisite for studying cytokine expression. Here phorbol 12-myristate 13-acetate (PMA)/ionomycin in vitro stimulation has been applied. In order to accumulate the cytokines within the cells, protein secretion needs to be inhibited, by the addition of reagents that inhibit protein secretion during the stimulation. The two most widely used reagents are monensin and brefeldin A (BFA). These reagents differ somewhat in their mode of action, which might explain their different effects. Monensin is an inhibitor of trans-Golgi function, while BFA inhibits protein transport between the endoplasmic reticulum (ER) and the Golgi. CD69, a very early activation marker on lymphocytes and neutrophils, was monitored in order to measure the efficacy of the protein secretion inhibition. Here we report that: (a) BFA, but not Monensin, is able to completely block extracellular CD69 expression on mice splenocytes after in vitro stimulation with PMA/ionomycin; (b) Monensin is more toxic than BFA and increases the relative amount of CD4+ cells due to a more profound increase in dead cells in the CD4- population; (c) CD69 is a useful marker when setting up intracellular staining of cytokines for flow cytometry.