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Blood Cancer Journal Jun 2018Therapy for acute myeloid leukemia (AML) involves intense cytotoxic treatment and yet approximately 70% of AML are refractory to initial therapy or eventually relapse....
Therapy for acute myeloid leukemia (AML) involves intense cytotoxic treatment and yet approximately 70% of AML are refractory to initial therapy or eventually relapse. This is at least partially driven by the chemo-resistant nature of the leukemic stem cells (LSCs) that sustain the disease, and therefore novel anti-LSC therapies could decrease relapses and improve survival. We performed in silico analysis of highly prognostic human AML LSC gene expression signatures using existing datasets of drug-gene interactions to identify compounds predicted to target LSC gene programs. Filtering against compounds that would inhibit a hematopoietic stem cell (HSC) gene signature resulted in a list of 151 anti-LSC candidates. Using a novel in vitro LSC assay, we screened 84 candidate compounds at multiple doses and confirmed 14 drugs that effectively eliminate human AML LSCs. Three drug families presenting with multiple hits, namely antihistamines (astemizole and terfenadine), cardiac glycosides (strophanthidin, digoxin and ouabain) and glucocorticoids (budesonide, halcinonide and mometasone), were validated for their activity against human primary AML samples. Our study demonstrates the efficacy of combining computational analysis of stem cell gene expression signatures with in vitro screening to identify novel compounds that target the therapy-resistant LSC at the root of relapse in AML.
Topics: Apoptosis; Biomarkers; Biomarkers, Tumor; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Computational Biology; Cytarabine; Drug Discovery; Drug Screening Assays, Antitumor; Gene Expression Profiling; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Neoplastic Stem Cells; Transcriptome
PubMed: 29921955
DOI: 10.1038/s41408-018-0087-2 -
Free Radical Biology & Medicine Jun 2018Nitric oxide (NO) is an essential signaling molecule in the body, regulating numerous biological processes. Beside its physiological roles, NO affects drug metabolism by...
Nitric oxide (NO) is an essential signaling molecule in the body, regulating numerous biological processes. Beside its physiological roles, NO affects drug metabolism by modulating the activity and/or expression of cytochrome P450 enzymes. Previously, our lab showed that NO generation caused by inflammatory stimuli results in CYP2B6 degradation via the ubiquitin-proteasome pathway. In the current study, we tested the NO-mediated regulation of CYP2J2 that metabolizes arachidonic acids to bioactive epoxyeicosatrienoic acids, as well as therapeutic drugs such as astemizole and ebastine. To investigate the effects of NO on CYP2J2 expression and activity, Huh7 cells stably transduced with CYP2J2 with a C-terminal V5 tag were treated with dipropylenetriamine-NONOate (DPTA), a NO donor. The level of CYP2J2 proteins were decreased in a time- and concentration-dependent manner, and the activity was also rapidly inhibited. However, mRNA expression was not altered and the protein synthesis inhibitor cycloheximide did not attenuate DPTA-mediated downregulation of CYP2J2. Removal of DPTA from the culture media quickly restored the activity of remaining CYP2J2, and no further CYP2J2 degradation occurred. To determine the mechanism of CYP2J2 down-regulation by NO, cells were treated with DPTA in the presence or absence of protease inhibitors including proteasomal, lysosomal and calpain inhibitors. Remarkably, the down-regulation of CYP2J2 by NO was attenuated by calpeptin, a calpain inhibitor. However, other calpain inhibitors or calcium chelator show no inhibitory effects on the degradation. The proteasome inhibitor bortezomib showed small but significant restoration of CYP2J2 levels although stimulated ubiquitination of CYP2J2 was not detected. In conclusion, these data suggest that NO regulates CYP2J2 posttranslationally and NO-evoked CYP2J2 degradation undergoes ubiquitin-independent proteasomal degradation pathway unlike CYP2B6.
Topics: Antineoplastic Agents; Bortezomib; Carcinoma, Hepatocellular; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Down-Regulation; Free Radical Scavengers; Gene Expression Regulation; Humans; Liver Neoplasms; Nitric Oxide; Nitric Oxide Donors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Tumor Cells, Cultured
PubMed: 29715548
DOI: 10.1016/j.freeradbiomed.2018.04.576 -
Scientific Reports Jan 2018Voltage-gated ion channels (VGCs) are prime targets for the pharmaceutical industry, but drug profiling on VGCs is challenging, since drug interactions are confined to...
Voltage-gated ion channels (VGCs) are prime targets for the pharmaceutical industry, but drug profiling on VGCs is challenging, since drug interactions are confined to specific conformational channel states mediated by changes in transmembrane potential. Here we combined various optogenetic tools to develop dynamic, high-throughput drug profiling assays with defined light-step protocols to interrogate VGC states on a millisecond timescale. We show that such light-induced electrophysiology (LiEp) yields high-quality pharmacological data with exceptional screening windows for drugs acting on the major cardiac VGCs, including hNa1.5, hK1.5 and hERG. LiEp-based screening remained robust when using a variety of optogenetic actuators (ChR2, ChR2(H134R), CatCh, ChR2-EYFP-βArchT) and different types of organic (RH421, Di-4-ANBDQPQ, BeRST1) or genetic voltage sensors (QuasAr1). The tractability of LiEp allows a versatile and precise alternative to state-of-the-art VGC drug screening platforms such as automated electrophysiology or FLIPR readers.
Topics: Astemizole; Dose-Response Relationship, Drug; ERG1 Potassium Channel; Flecainide; Gene Expression; HEK293 Cells; High-Throughput Screening Assays; Humans; Lidocaine; Membrane Potentials; NAV1.5 Voltage-Gated Sodium Channel; Optogenetics; Patch-Clamp Techniques; Phosphines; Plasmids; Potassium Channel Blockers; Quinidine; Voltage-Gated Sodium Channel Blockers
PubMed: 29348631
DOI: 10.1038/s41598-018-19412-z -
OncoTargets and Therapy 2017Lung cancer is a major cause of cancer mortality. Thus, novel therapies are urgently needed. Repositioning of old drugs is gaining great interest in cancer treatment....
Lung cancer is a major cause of cancer mortality. Thus, novel therapies are urgently needed. Repositioning of old drugs is gaining great interest in cancer treatment. Astemizole is an antihistamine proposed to be repositioned for cancer therapy. This drug targets several molecules involved in cancer including histamine receptors, ABC transporters and the potassium channels Eag1 and HERG. Astemizole inhibits the proliferation of different cancer cells including those from cervix, breast, leukemia and liver. Gefitinib is widely used to treat lung cancer; however, no response or drug resistance occurs in many cases. Here, we studied the combined effect of astemizole and gefitinib on the proliferation, survival, apoptosis and gene and protein expression of Eag1 channels in the human lung cancer cell lines A549 and NCI-H1975. Cell proliferation and survival were studied by the MTT method and the colony formation assay, respectively; apoptosis was investigated by flow cytometry. Gene expression was assessed by real-time polymerase chain reaction (RT-PCR), and protein expression was studied by Western blot analysis and immunocytochemistry. We obtained the inhibitory concentrations 20 and 50 (IC and IC, respectively) values for each drug from the cell proliferation experiments. Drug combination at their IC had a superior effect by reducing cell proliferation and survival in up to 80% and 100%, respectively. The drugs alone did not affect apoptosis of H1975 cells, but the drug combination at their IC increased apoptosis roughly four times in comparison to the effect of the drugs alone. Eag1 mRNA levels and protein expression were decreased by the drug combination in A549 cells, and astemizole induced subcellular localization changes of the channel protein in these cells. Our in vitro studies strongly suggest that the combination astemizole-gefitinib may be a novel and promising therapy for lung cancer patients.
PubMed: 29263676
DOI: 10.2147/OTT.S144506 -
The Journal of Toxicological Sciences 2017Monitoring dramatic changes in intracellular calcium ion levels during cardiac contraction and relaxation, known as calcium transient, in human induced pluripotent stem...
Monitoring dramatic changes in intracellular calcium ion levels during cardiac contraction and relaxation, known as calcium transient, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be an attractive strategy for assessing compounds on cardiac contractility. In addition, as arrhythmogenic compounds are known to induce characteristic waveform changes in hiPSC-CMs, it is expected that calcium transient would allow evaluation of not only compound-induced effects on cardiac contractility, but also compound arrhythmogenic potential. Using a combination of calcium transient in hiPSC-CMs and a fast kinetic fluorescence imaging detection system, we examined in this study changes in calcium transient waveforms induced by a series of 17 compounds that include positive/negative inotropic agents as well as cardiac ion channel activators/inhibitors. We found that all positive inotropic compounds induced an increase in peak frequency and/or peak amplitude. The effects of a negative inotropic compound could clearly be detected in the presence of a β-adrenergic receptor agonist. Furthermore, most arrhythmogenic compounds raised the ratio of peak decay time to peak rise time (D/R ratio) in calcium transient waveforms. Compound concentrations at which these parameters exceeded cutoff values correlated well with systemic exposure levels at which arrhythmias were reported to be evoked. In conclusion, we believe that peak analysis of calcium transient and determination of D/R ratio are reliable methods for assessing compounds' cardiac contractility and arrhythmogenic potential, respectively. Using these approaches would allow selection of compounds with low cardiotoxic potential at the early stage of drug discovery.
Topics: Arrhythmias, Cardiac; Astemizole; Calcium; Calcium Channel Blockers; Cardiotonic Agents; Cell Differentiation; Cells, Cultured; Digoxin; Dose-Response Relationship, Drug; Drug Discovery; Fluoroquinolones; Induced Pluripotent Stem Cells; Isoproterenol; Moxifloxacin; Myocardial Contraction; Myocytes, Cardiac; Propranolol; Toxicity Tests; Verapamil
PubMed: 28717111
DOI: 10.2131/jts.42.519 -
Molecules (Basel, Switzerland) May 2017Oxidative damage can lead to a wide range of diseases. Nrf2 is an important transcription factor that regulates many of the cytoprotective enzymes involved in the...
Oxidative damage can lead to a wide range of diseases. Nrf2 is an important transcription factor that regulates many of the cytoprotective enzymes involved in the oxidative stress response. Therefore, targeting the regulation of Nrf2 activation is one logical and effective strategy to prevent or lower the risk of oxidative stress-related diseases. Until now, most research has focused on electrophilic indirect Nrf2 activators, but the risk of 'off-target' effects may be associated with these activators. To find novel small non-electrophilic modulators of Nrf2, we started from chemical agents derived from a connectivity map (cMap) and identified 22 non-electrophilic potential Nrf2-activating drugs through a drug repositioning tactic. By determining the expression changes of antioxidant genes in MCF7 cells that were treated with the potential Nrf2 activators using quantitative real-time polymerase chain reaction RT-PCR (real-time polymerase chain reaction) (qRT-PCR), astemizole was found to have a greater scale of upregulating antioxidant genes , , and than the positive control d,l-sulforaphane, although the testing concentration was lower than that of the control. Astemizole is a good potential redox regulator and deserves more pharmacodynamic experimentation to test and verify its feasibility for use as an Nrf2 activator.
Topics: Antioxidants; Cell Line, Tumor; Drug Discovery; Drug Evaluation, Preclinical; Drug Repositioning; Gene Expression Regulation; Humans; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress
PubMed: 28587109
DOI: 10.3390/molecules22060883 -
A structure- and chemical genomics-based approach for repositioning of drugs against VCP/p97 ATPase.Scientific Reports Mar 2017Valosin-containing protein (VCP/p97) ATPase (a.k.a. Cdc48) is a key member of the ER-associated protein degradation (ERAD) pathway. ERAD and VCP/p97 have been implicated...
Valosin-containing protein (VCP/p97) ATPase (a.k.a. Cdc48) is a key member of the ER-associated protein degradation (ERAD) pathway. ERAD and VCP/p97 have been implicated in a multitude of human diseases, such as neurodegenerative diseases and cancer. Inhibition of VCP/p97 induces proteotoxic ER stress and cell death in cancer cells, making it an attractive target for cancer treatment. However, no drugs exist against this protein in the market. Repositioning of drugs towards new indications is an attractive alternative to the de novo drug development due to the potential for significantly shorter time to clinical translation. Here, we employed an integrative strategy for the repositioning of drugs as novel inhibitors of the VCP/p97 ATPase. We integrated structure-based virtual screening with the chemical genomics analysis of drug molecular signatures, and identified several candidate inhibitors of VCP/p97 ATPase. Importantly, experimental validation with cell-based and in vitro ATPase assays confirmed three (ebastine, astemizole and clotrimazole) out of seven tested candidates (~40% true hit rate) as direct inhibitors of VCP/p97 and ERAD. This study introduces an effective integrative strategy for drug repositioning, and identified new drugs against the VCP/p97/ERAD pathway in human diseases.
Topics: Allosteric Regulation; Binding Sites; Drug Discovery; Drug Repositioning; Enzyme Inhibitors; Genomics; Humans; Inhibitory Concentration 50; Models, Molecular; Molecular Conformation; Protein Binding; Reproducibility of Results; Structure-Activity Relationship; Valosin Containing Protein
PubMed: 28322292
DOI: 10.1038/srep44912 -
Proceedings of the National Academy of... Apr 2017Prion diseases, like Alzheimer's disease and Parkinson disease, are rapidly progressive neurodegenerative disorders caused by misfolding followed by aggregation and...
Prion diseases, like Alzheimer's disease and Parkinson disease, are rapidly progressive neurodegenerative disorders caused by misfolding followed by aggregation and accumulation of protein deposits in neuronal cells. Here we measure intramolecular polypeptide backbone reconfiguration as a way to understand the molecular basis of prion aggregation. Our hypothesis is that when reconfiguration is either much faster or much slower than bimolecular diffusion, biomolecular association is not stable, but as the reconfiguration rate becomes similar to the rate of biomolecular diffusion, the association is more stable and subsequent aggregation is faster. Using the technique of Trp-Cys contact quenching, we investigate the effects of various conditions on reconfiguration dynamics of the Syrian hamster and rabbit prion proteins. This protein exhibits behavior in all three reconfiguration regimes. We conclude that the hamster prion is prone to aggregation at pH 4.4 because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs, whereas the rabbit sequence avoids aggregation by reconfiguring 10 times faster than the hamster sequence.
Topics: Animals; Diffusion; Hydrophobic and Hydrophilic Interactions; Mesocricetus; Models, Molecular; Prion Proteins; Protein Aggregates; Protein Conformation; Protein Unfolding; Rabbits
PubMed: 28320943
DOI: 10.1073/pnas.1620400114 -
Molecular Medicine Reports Apr 2017The loss of dopaminergic neurons and the resultant motor impairment are hallmarks of Parkinson's disease. The SH‑SY5Y cell line is a model of dopaminergic neurons, and...
The loss of dopaminergic neurons and the resultant motor impairment are hallmarks of Parkinson's disease. The SH‑SY5Y cell line is a model of dopaminergic neurons, and allows for the study of dopaminergic neuronal injury. Previous studies have revealed changes in Ether à go‑go 1 (Eag1) potassium channel expression during p53-induced SH‑SY5Y apoptosis, and the regulatory involvement of microRNA‑34a (miR‑34a) was demonstrated. In the present study, the involvement of Eag1 and miR‑34a in rotenone‑induced SH‑SY5Y cell injury was investigated. Rotenone is a neurotoxin, which is often used to generate models of Parkinson's disease, since it causes the death of nigrostriatal neurons by inducing intracellular aggregation of alpha synuclein and ubiquitin. In the present study, rotenone resulted in a dose‑dependent decrease in cell viability, as revealed by 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) and trypan blue cell counting assays. In addition, Eag1 was demonstrated to be constitutively expressed by SH‑SY5Y cells, and involved in cell viability. Suppression of Eag1 with astemizole resulted in a dose‑dependent decrease in cell viability, as revealed by MTT assay. Astemizole also enhanced the severity of rotenone‑induced injury in SH‑SY5Y cells. RNA interference against Eag1, using synthetic small interfering RNAs (siRNAs), corroborated this finding, as siRNAs potentiated rotenone‑induced injury. Eag1‑targeted siRNAs (kv10.1‑3 or EAG1hum_287) resulted in a statistically significant 16.4‑23.5% increase in vulnerability to rotenone. An increased number of apoptotic nuclei were observed in cells transfected with EAG1hum_287. Notably, this siRNA intensified rotenone‑induced apoptosis, as revealed by an increase in caspase 3/7 activity. Conversely, a miR‑34a inhibitor was demonstrated to exert neuroprotective effects. The viability of cells exposed to rotenone for 24 or 48 h and treated with miR‑34a inhibitor was restored by 8.4‑8.8%. In conclusion, Eag1 potassium channels and miR‑34a are involved in the response to rotenone-induced injury in SH‑SY5Y cells. The neuroprotective effect of mir‑34a inhibitors merits further investigations in animal models of Parkinson's disease.
Topics: Astemizole; Cell Death; Cell Line, Tumor; Cell Survival; Dopaminergic Neurons; Ether-A-Go-Go Potassium Channels; Gene Silencing; Humans; Immunohistochemistry; MicroRNAs; RNA, Small Interfering; Rotenone; Transfection
PubMed: 28259991
DOI: 10.3892/mmr.2017.6191 -
Biologics : Targets & Therapy 2016Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide. HCC is usually asymptomatic at potential curative stages, and it has very poor prognosis if... (Review)
Review
Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide. HCC is usually asymptomatic at potential curative stages, and it has very poor prognosis if detected later. Thus, the identification of early biomarkers and novel therapies is essential to improve HCC patient survival. Ion channels have been proposed as potential tumor markers and therapeutic targets for several cancers including HCC. Especially, the ether à-go-go-1 (Eag1) voltage-gated potassium channel has been suggested as an early marker for HCC. Eag1 is overexpressed during HCC development from the cirrhotic and the preneoplastic lesions preceding HCC in a rat model. The channel is also overexpressed in human HCC. Astemizole has gained great interest as a potential anticancer drug because it targets several proteins involved in cancer including Eag1. Actually, in vivo studies have shown that astemizole may have clinical utility for HCC prevention and treatment. Here, we will review first some general aspects of HCC including the current biomarkers and therapies, and then we will focus on Eag1 channels as promising tools in the early diagnosis of HCC.
PubMed: 27703327
DOI: 10.2147/BTT.S87402