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International Journal of Cancer Nov 1998ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol...
ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3-kinase (PI3-kinase). In order to determine the contribution of ATM and DNA-PKcs to the increased sensitivity of cells to DNA-damaging agents observed in the presence of PI3-kinase inhibitors, we examined the effects of a PI3-kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X-irradiation and ultraviolet (UV)-irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X-irradiation, but not to MMC or UV-irradiation. Hypersensitivity to BLM and X-irradiation and normal sensitivity to MMC and UV-irradiation are characteristic phenotypes of both AT and scid mice. DNA-dependent protein kinase (DNA-PK) activity was suppressed by wortmannin to 45-65% of the control values in all of the cells except SCF, in which DNA-PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X-irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA-PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy.
Topics: Androstadienes; Animals; Antibiotics, Antineoplastic; Ataxia Telangiectasia Mutated Proteins; Bleomycin; Cell Cycle Proteins; DNA; DNA-Activated Protein Kinase; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Mice; Mice, SCID; Nuclear Proteins; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proteins; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wortmannin
PubMed: 9808536
DOI: 10.1002/(sici)1097-0215(19981123)78:5<642::aid-ijc19>3.0.co;2-3 -
Molecular Biology of the Cell Mar 1996Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin...
Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.
Topics: Androstadienes; Down-Regulation; Endocytosis; Endosomes; HeLa Cells; Humans; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Transferrin; Tumor Cells, Cultured; Wortmannin
PubMed: 8868465
DOI: 10.1091/mbc.7.3.355 -
Journal of Experimental Botany 2009Wortmannin, a specific inhibitor of phosphatidyl-inositol 3-kinase, is a useful tool for studying protein trafficking and identifying organelles in the plant secretory...
Wortmannin, a specific inhibitor of phosphatidyl-inositol 3-kinase, is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. It has recently been demonstrated that wortmannin at 16.5 microM or 33 microM caused the prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs) by their enrichment in vacuolar sorting receptor (VSRs) proteins and the BP-80 reporter, to form small vacuoles rapidly. However, the source(s) of the membrane needed for the rapid enlargement of PVCs/MVBs has been unclear. Using both confocal immunofluorescence and immunogold EM with high pressure freeze substitution of plant samples, it has been demonstrated here that wortmannin induces homotypic fusions of PVCs/MVBs thus providing an explanation for the demand for extra membrane. In addition, possible wortmannin-induced fusions between the trans-Golgi network (TGN) and PVC, as well as between the small internal vesicles and PVC membrane, were also observed and they may also contribute to the membranes needed for PVC enlargement. In contrast to mammalian cells and yeast, wortmannin-induced fusion of PVCs appears to be unique to plants.
Topics: Androstadienes; Cell Line; Endosomes; Membrane Fusion; Nicotiana; Vacuoles; Wortmannin
PubMed: 19436047
DOI: 10.1093/jxb/erp136 -
The Journal of Biological Chemistry May 1998Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A...
Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A reliable means to introduce these lipids into intact cells would be of great value for showing the physiological roles of this pathway and for testing the specificity of pharmacological inhibitors of the kinases. We have stereospecifically synthesized di-C8-PIP3/AM and di-C12-PIP3/AM, the heptakis(acetoxymethyl) esters of dioctanoyl- and dilauroylphosphatidylinositol 3,4,5-trisphosphate, in 14 steps from myo-inositol. The ability of these uncharged lipophilic derivatives to deliver phosphatidylinositol 3,4,5-trisphosphate across cell membranes was demonstrated on 3T3-L1 adipocytes and T84 colon carcinoma monolayers. Insulin stimulation of hexose uptake into adipocytes was inhibited by the kinase inhibitor wortmannin and was largely restored by di-C8-PIP3/AM, which had no effect in the absence of insulin. Thus phosphatidylinositol 3,4,5-trisphosphate or a metabolite was necessary but not sufficient for stimulation of hexose transport. In T84 epithelial monolayers, di-C12-PIP3/AM mimicked epidermal growth factor in inhibiting chloride secretion and potassium efflux, suggesting that phosphatidylinositol 3,4, 5-trisphosphate was sufficient to modulate these fluxes and mediate epidermal growth factor's action.
Topics: 3T3 Cells; Adipocytes; Androstadienes; Animals; Cell Membrane Permeability; Epidermal Growth Factor; Esters; Hexoses; Insulin; Magnetic Resonance Spectroscopy; Mice; Phosphatidylinositol Phosphates; Tumor Cells, Cultured; Wortmannin
PubMed: 9556583
DOI: 10.1074/jbc.273.18.11017 -
PLoS Computational Biology Dec 2018An expanded chemical space is essential for improved identification of small molecules for emerging therapeutic targets. However, the identification of targets for novel...
An expanded chemical space is essential for improved identification of small molecules for emerging therapeutic targets. However, the identification of targets for novel compounds is biased towards the synthesis of known scaffolds that bind familiar protein families, limiting the exploration of chemical space. To change this paradigm, we validated a new pipeline that identifies small molecule-protein interactions and works even for compounds lacking similarity to known drugs. Based on differential mRNA profiles in multiple cell types exposed to drugs and in which gene knockdowns (KD) were conducted, we showed that drugs induce gene regulatory networks that correlate with those produced after silencing protein-coding genes. Next, we applied supervised machine learning to exploit drug-KD signature correlations and enriched our predictions using an orthogonal structure-based screen. As a proof-of-principle for this regimen, top-10/top-100 target prediction accuracies of 26% and 41%, respectively, were achieved on a validation of set 152 FDA-approved drugs and 3104 potential targets. We then predicted targets for 1680 compounds and validated chemical interactors with four targets that have proven difficult to chemically modulate, including non-covalent inhibitors of HRAS and KRAS. Importantly, drug-target interactions manifest as gene expression correlations between drug treatment and both target gene KD and KD of genes that act up- or down-stream of the target, even for relatively weak binders. These correlations provide new insights on the cellular response of disrupting protein interactions and highlight the complex genetic phenotypes of drug treatment. With further refinement, our pipeline may accelerate the identification and development of novel chemical classes by screening compound-target interactions.
Topics: Cell Line; Computational Biology; Computer Simulation; Databases, Nucleic Acid; Drug Discovery; Drug Evaluation, Preclinical; Gene Expression Profiling; Gene Knockdown Techniques; Gene Ontology; Gene Regulatory Networks; Humans; Models, Molecular; Molecular Docking Simulation; Protein Kinase Inhibitors; Proteins; Ubiquitin-Protein Ligases; Wortmannin; ras Proteins
PubMed: 30532261
DOI: 10.1371/journal.pcbi.1006651 -
Journal of Radiation Research Mar 2020Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase...
Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.
Topics: Animals; Cell Line; Cell Survival; DNA Breaks, Double-Stranded; Histones; Humans; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Kinase Inhibitors; Radiation Tolerance; Wortmannin
PubMed: 32052028
DOI: 10.1093/jrr/rrz102 -
International Journal of Pharmaceutics Jan 2019Human organic anion transporter 4 (hOAT4) belongs to a class of organic anion transporters that exert critical function in the secretion, absorption, and distribution of...
Human organic anion transporter 4 (hOAT4) belongs to a class of organic anion transporters that exert critical function in the secretion, absorption, and distribution of numerous drugs in the body, such as anti-viral drugs, anti-cancer therapeutics, antibiotics, antihypertensive medicine, and anti-inflammatory drugs. hOAT4 is richly existent in the kidney and placenta. We previously established that serum- and glucocorticoid-inducible kinases (sgk) stimulate hOAT4 expression and transport activity by abrogating the inhibitory effect of a ubiquitin ligase Nedd4-2. Insulin is one of the upstream signaling molecules for sgk. We therefore investigated the effect of insulin on hOAT4 function. We showed that insulin stimulated hOAT4 expression and transport activity, and the action of insulin was abolished in cells overexpressing Nedd4-2-specific siRNA to knockdown the endogenous Nedd4-2. We further showed that insulin phosphorylated serine 327 on Nedd4-2 and weakened the interaction between hOAT4 and Nedd4-2. Interestingly, in cells overexpressing sgk2, the stimulatory effect of insulin on hOAT4 was diminished. In addition, the stimulatory effect of insulin on hOAT4 was blocked by wortmannin and buparlisib, two PI3K inhibitors. In conclusion, our study demonstrated that insulin stimulates hOAT4 expression and transport activity by abrogating the inhibition effect of Nedd4-2 on the transporter. Moreover, insulin regulates hOAT4 by competing with sgk2 rather than through sgk2.
Topics: Aminopyridines; Animals; COS Cells; Chlorocebus aethiops; Gene Knockdown Techniques; Humans; Immediate-Early Proteins; Insulin; Morpholines; Nedd4 Ubiquitin Protein Ligases; Organic Anion Transporters, Sodium-Independent; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; RNA, Small Interfering; Wortmannin
PubMed: 30453017
DOI: 10.1016/j.ijpharm.2018.11.040 -
The Journal of Biological Chemistry Oct 1995Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that... (Comparative Study)
Comparative Study
Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase.
Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that these compounds are not as selective as assumed and demonstrate inhibition of bombesin-stimulated phospholipase A2 activity by both wortmannin and DMV with an IC50 (2 nM) which is slightly more potent than the inhibition of insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate generation in these cells (approximately 10nM). While it has not been possible to fully block in vitro phospholipase A2 activity with wortmannin, inhibition cannot be a consequence of inhibition of PI 3-kinase activity since bombesin fails to generate 3-phosphorylated lipids in the intact cell. Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is not as specific as previously reported, and experimental conclusions based solely on its use should be treated with caution.
Topics: 3T3 Cells; Androstadienes; Androstenes; Animals; Bombesin; Dose-Response Relationship, Drug; Enzyme Activation; Mice; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphodiesterase Inhibitors; Phospholipase D; Phospholipases; Phospholipases A; Phospholipases A2; Phosphotransferases (Alcohol Group Acceptor); Type C Phospholipases; Wortmannin
PubMed: 7592698
DOI: 10.1074/jbc.270.43.25352 -
Cellular Physiology and Biochemistry :... 2018It has been implicated that calcium supplementation is involved in reducing body weight/fat and improving glucose homeostasis. However, the underlying mechanisms are...
Calcium Supplementation Enhanced Adipogenesis and Improved Glucose Homeostasis Through Activation of Camkii and PI3K/Akt Signaling Pathway in Porcine Bone Marrow Mesenchymal Stem Cells (pBMSCs) and Mice Fed High Fat Diet (HFD).
BACKGROUND/AIMS
It has been implicated that calcium supplementation is involved in reducing body weight/fat and improving glucose homeostasis. However, the underlying mechanisms are still not fully understood. Here, we investigated the effects of calcium supplementation on adipogenesis and glucose homeostasis in porcine bone marrow mesenchymal stem cells (pBMSCs) and high fat diet (HFD)-fed mice and explored the involved signaling pathways.
METHODS
In vitro, pBMSCs were treated with 4 mM extracellular calcium ([Ca2+]o) and/or 1 μM nifedipine, 0.1 μM BAPTA-AM, 1 μM KN-93, 50 nM wortmannin for 10 days. The intracellular calcium ([Ca2+]i) levels were measured using Fluo 3-AM by flow cytometry. The adipogenic differentiation of pBMSCs was determined by Oil Red-O staining and triglyceride assay. The expression of marker genes involved in adipogenesis (peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα)) and glucose uptake (glucose transporter 4 (GLUT4)), as well as the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and PI3K/Akt-FoxO1/AS160 signaling pathways were determined by Western blotting. Glucose uptake and utilization were examined using 2-NBDG assay and glucose content assay, respectively. In vivo, C57BL/6J male mice were fed a HFD (containing 1.2% calcium) without or with 0.6% (w/w) calcium chloride in drinking water for 13 weeks. The adipogenesis, glucose homeostasis and the involvement of CaMKII and PI3K/Akt signaling pathway were also assessed.
RESULTS
In vitro, [Ca2+]o stimulated pBMSCs adipogenesis by increasing [Ca2+]i level and activating CaMKII and PI3K/Akt-FoxO1 pathways. In addition, [Ca2+]o promoted glucose uptake/utilization by enhancing AS160 phosphorylation, GLUT4 expression and translocation. However, the stimulating effects of [Ca2+]o on pBMSCs adipogenesis and glucose uptake/utilization were abolished by L-VGCC blocker Nifedipine, [Ca2+]i chelator BAPTA-AM, CaMKII inhibitor KN-93, or PI3K inhibitor Wortmannin. In vivo, calcium supplementation decreased body weight and fat content, increased adipocyte number, and improved glucose homeostasis, with elevated PPARγ and GLUT4 expression and PI3K/Akt activation in iWAT.
CONCLUSION
calcium supplementation enhanced adipogenesis and glucose uptake in pBMSCs, which was coincident with the increased adipocyte number and improved glucose homeostasis in HFD-fed mice, and was associated with activation of CaMKII and PI3K/Akt-FoxO1/AS160 pathways. These data provided a broader understanding of the mechanisms underlying calcium-induced body weight/fat loss and glycemic control.
Topics: Adipogenesis; Animals; Bone Marrow Cells; Calcium; Calcium Channels, L-Type; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Differentiation; Diet, High-Fat; Glucose; Glucose Transporter Type 4; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; PPAR gamma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Swine; Wortmannin
PubMed: 30439703
DOI: 10.1159/000495171 -
Molecular Plant Jun 2014Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein...
Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein VTI11 in homotypic fusion of protein storage and lytic vacuoles. Fusion of the multiple vacuoles present in vti11 mutants could be induced by treatment with Wortmannin and LY294002, which are inhibitors of Phosphatidylinositol 3-Kinase (PI3K). We provide evidence that Phosphatidylinositol 3-Phosphate (PtdIns(3)P) regulates vacuole fusion in vti11 mutants, and that fusion of these vacuoles requires intact microtubules and actin filaments. Finally, we show that Wortmannin also induced the fusion of guard cell vacuoles in fava beans, where vacuoles are naturally fragmented after ABA-induced stomata closure. These results suggest a ubiquitous role of phosphoinositides in vacuole fusion, both during the development of the large central vacuole and during the dynamic vacuole remodeling that occurs as part of stomata movements.
Topics: Androstadienes; Arabidopsis; Arabidopsis Proteins; Chromones; Morpholines; Phosphatidylinositol Phosphates; Phosphoinositide-3 Kinase Inhibitors; Qb-SNARE Proteins; Vacuoles; Wortmannin
PubMed: 24569132
DOI: 10.1093/mp/ssu019