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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 -
Drug Metabolism and Pharmacokinetics Jun 2018Cytochrome P450 2J2 (CYP2J2) is involved in the metabolism of drugs, including albendazole, astemizole, ebastine, and endogenous substrates. In a previous study, we used...
Cytochrome P450 2J2 (CYP2J2) is involved in the metabolism of drugs, including albendazole, astemizole, ebastine, and endogenous substrates. In a previous study, we used recombinant CYP2J2 and determined whether danazol, hydroxyebastine, telmisartan, and terfenadone inhibited CYP2J2 by using four representative CYP2J2 substrates, namely albendazole, astemizole, ebastine, and terfenadine. In this study, we evaluated the inhibitory potential of these four chemicals on human liver and intestinal microsomes, which are commonly used in a reaction phenotyping study. Among the four CYP2J2 inhibitors tested, terfenadone was strongest inhibitor of CYP2J2-mediated metabolism of albendazole, astemizole, and terfenadine with IC values of 0.31, 0.15, and 2.11 μM, respectively, in human liver microsomes (HLMs). In addition, terfenadone had strong inhibitory effect on the metabolism of the abovementioned drugs in human intestinal microsomes (HIMs), with IC values of 0.43, 0.08 and 1.07 μM, respectively. Danazol, weakly inhibited CYP2J2-mediated metabolism of albendazole and astemizole with IC values of 13.8 and 18.3 μM, respectively in HLMs, whereas it strongly inhibited the CYP2J2-mediated ebastine hydroxylase activity in HLMs and HIMs (IC = 1.93-1.95 μM). Our data suggest that terfenadone may be used as a general CYP2J2 inhibitor in reaction phenotyping study using HLMs and HIMs regardless of the substrate used.
Topics: Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Humans; Intestinal Mucosa; Intestines; Liver; Microsomes; Structure-Activity Relationship; Terfenadine
PubMed: 29759885
DOI: 10.1016/j.dmpk.2018.02.001 -
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 -
Chemico-biological Interactions Jun 2018The use of Chinese herbal medicines and natural products has become increasingly popular in both China and Western societies as an alternative medicine for the treatment...
The use of Chinese herbal medicines and natural products has become increasingly popular in both China and Western societies as an alternative medicine for the treatment of diseases or as a health supplement. Danshen, the dried root of Salvia miltiorrhiza (Fam.Labiatae), which is rich in phenolic acids and tanshinones, is a widely used herbal medicine for the treatment of cardio-cerebrovascular diseases. The goal of this study was to examine the inhibitory effects of fifteen components derived from Danshen on CYP2C8 and CYP2J2, which are expressed both in human liver and cardiovascular systems. Recombinant CYP2C8 and CYP2J2 were used, and the mechanism, kinetics, and type of inhibition were determined. Taxol 6-hydroxylation and astemizole O-desmethyastemizole were determined as probe activities for CYP2C8 and CYP2J2, respectively. Metabolites formations were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that salvianolic acid A was a competitive inhibitor of CYP2C8 (K = 2.5 μM) and mixed-type inhibitor of CYP2J2 (K = 7.44 μM). Salvianolic acid C had moderate noncompetitive and mixed-type inhibitions on CYP2C8 (K = 4.82 μM) and CYP2J2 (K = 5.75 μM), respectively. Tanshinone IIA was a moderate competitive inhibitor of CYP2C8 (K = 1.18 μM). Dihydrotanshinone I had moderate noncompetitive inhibition on CYP2J2 (K = 6.59 μM), but mechanism-based inhibition on CYP2C8 (K = 0.43 μM, k = 0.097 min). Tanshinone I was a moderate competitive inhibitor of CYP2C8 (K = 4.20 μM). These findings suggested that Danshen preparations appear not likely to pose a significant risk of drug interactions mediated by CYP2C8 after oral administration; but their inhibitory effects on intestinal CYP2J2 mediated drug metabolism should not be neglected when they are given orally in combination with other drugs. Additionally, this study provided novel insights into the underling pharmacological mechanisms of Danshen components from the perspective of CYP2C8 and CYP2J2 inhibition.
Topics: Cytochrome P-450 CYP2C8; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drugs, Chinese Herbal; Humans; Inhibitory Concentration 50; Kinetics; Recombinant Proteins; Salvia miltiorrhiza; Taxoids; Time Factors
PubMed: 29689254
DOI: 10.1016/j.cbi.2018.04.011 -
Phytomedicine : International Journal... Mar 2018Broussonetia papyrifera (L.) Ventenat, a traditional medicinal herb, has been applied as a folk medicine to treat various diseases. Broussochalcone A (BCA), a chalcone...
BACKGROUND
Broussonetia papyrifera (L.) Ventenat, a traditional medicinal herb, has been applied as a folk medicine to treat various diseases. Broussochalcone A (BCA), a chalcone compound isolated from the cortex of Broussonetia papyrifera (L.) Ventenat, exhibits several biological activities including potent anti-oxidant, antiplatelet, and cytotoxic effects.
PURPOSE
The purpose of this study is to elucidate the inhibitory effect of BCA against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines.
STUDY DESIGN
The inhibitory effect of BCA on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its anti-cancer effect against human hepatoma HepG2 cells was also evaluated.
METHODS
Two representative CYP2J2-specific probe substrates, astemizole and ebastine, were incubated in HLMs with BCA. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. To investigate the binding model between BCA and CYP2J2, we carried out structure-based docking simulations by using software and scripts written in-house.
RESULTS
BCA inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities in a concentration dependent manner with K values of 2.3 and 3.7 µM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner with activation of apoptosis related proteins.
CONCLUSION
Overall, this was the first report of the inhibitory effects of BCA on CYP2J2 in HLMs. The present data suggest that BCA is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities.
Topics: Antineoplastic Agents, Phytogenic; Astemizole; Butyrophenones; Cell Proliferation; Chalcones; Chromatography, Liquid; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Forkhead Box Protein O3; Hep G2 Cells; Humans; Microsomes, Liver; Molecular Docking Simulation; Piperidines; Resorcinols; Tandem Mass Spectrometry
PubMed: 29655687
DOI: 10.1016/j.phymed.2018.03.032 -
Phytomedicine : International Journal... Jan 2018Cytochrome P450 2J2 (CYP2J2) is not only highly expressed in many kinds of human tumors, but also promotes tumor cell growth via regulating the metabolism of arachidonic...
BACKGROUND
Cytochrome P450 2J2 (CYP2J2) is not only highly expressed in many kinds of human tumors, but also promotes tumor cell growth via regulating the metabolism of arachidonic acids. CYP2J2 inhibitors can significantly reduce proliferation, migration and promote apoptosis of tumor cells by inhibiting epoxyeicosatrienoic acids (EETs) biosynthesis. Therefore screening CYP2J2 inhibitors is a significant way for the development of anti-cancer drug.
PURPOSE
The aim of this study was to identify a new CYP2J2 inhibitor from fifty natural compounds obtained from plants.
STUDY DESIGN
CYP2J2 inhibitor was screened from a natural compounds library and further the inhibitory manner and mechanism were evaluated. Its cytotoxicity against HepG2 and SMMC-7721 cell lines was also estimated.
METHODS
The inhibitory effect was evaluated in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant CYP2J2 (rCYP2J2), using astemizole as a probe substrate and inhibitory mechanism was illustrated through molecular docking. The cytotoxicity was detected using SRB.
RESULTS
In all candidates, plumbagin showed the strongest inhibitory effect on the CYP2J2-mediated astemizole O-demethylation activity. Further study revealed that plumbagin potently inhibited CYP2J2 activity with IC value at 3.82 µM, 3.37 µM and 1.17 µM in RLMs, HLMs and rCYP2J2, respectively. Enzyme kinetic studies showed that plumbagin was a mixed-type inhibitor of CYP2J2 in HLMs and rCYP2J2 with K value of 1.88 µM and 0.92 µM, respectively. Docking data presented that plumbagin interacted with CYP2J2 mainly through GLU 222 and ALA 223. Moreover, plumbagin showed strongly cytotoxic effects on hepatoma cell lines, such as HepG2 and SMMC-7721, with lower toxicity on rat primary hepatocytes. Plumbagin had no effect on the protein expression of CYP2J2 in HepG2 and SMMC-7721, while down-regulated the mRNA level of anti-apoptosis protein Bcl-2.
CONCLUSION
This study found out a new CYP2J2 inhibitor plumbagin from fifty natural compounds. Plumbagin presented a potential of anti-cancer pharmacological activity.
Topics: Animals; Antineoplastic Agents; Biological Products; Carcinoma, Hepatocellular; Cell Proliferation; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Evaluation, Preclinical; Hepatocytes; Humans; Kinetics; Liver Neoplasms; Male; Microsomes, Liver; Molecular Docking Simulation; Naphthoquinones; Rats, Sprague-Dawley
PubMed: 29433675
DOI: 10.1016/j.phymed.2017.12.026 -
Pharmacotherapy Mar 2018Use of the QT interval corrected for heart rate (QTc) on the electrocardiogram (ECG) to predict torsades de pointes (TdP) risk from culprit drugs is neither sensitive... (Meta-Analysis)
Meta-Analysis
BACKGROUND
Use of the QT interval corrected for heart rate (QTc) on the electrocardiogram (ECG) to predict torsades de pointes (TdP) risk from culprit drugs is neither sensitive nor specific. The ratio of the half-maximum inhibitory concentration of the hERG channel (hERG IC50) to the peak serum concentration of unbound drug (C ) is used during drug development to screen out chemical entities likely to cause TdP.
PURPOSE
To validate the use of the hERG IC50:C ratio to predict TdP risk from a culprit drug by its correlation with TdP incidence.
DATA SOURCES
Medline (between 1966 and March 2017) was accessed for hERG IC50 and C values from the antihistamine, fluoroquinolone, and antipsychotic classes to identify cases of drug-induced TdP. Exposure to a culprit drug was estimated from annual revenues reported by the manufacturer.
STUDY SELECTION
Inclusion criteria for TdP cases were provision of an ECG tracing that demonstrated QTc prolongation with TdP and normal serum values of potassium, calcium, and magnesium. Cases reported in patients with a prior rhythm disturbance and those involving a drug interaction were excluded.
DATA EXTRACTION AND SYNTHESIS
The Meta-Analysis of Observational Studies in Epidemiology checklist was used for epidemiological data extraction by two authors.
MAIN OUTCOME AND MEASURE
Negligible risk drugs were defined by an hERG IC50:C ratio that correlated with less than a 5% chance of one TdP event for every 100 million exposures (relative risk [RR] 1.0).
RESULTS
The hERG IC50:C ratio correlated with TdP risk (0.312; 95% confidence interval 0.205-0.476, p<0.0001), a ratio of 80 (RR 1.0). The RR from olanzapine is on par with loratadine; ziprasidone is comparable with ciprofloxacin. Drugs with an RR greater than 50 include astemizole, risperidone, haloperidol, and thioridazine.
CONCLUSIONS
The hERG IC50:C ratio was correlated with TdP incidence for culprit drugs. This validation provides support for the potential use of the hERG IC50:C ratio for clinical decision making in instances of drug selection where TdP risk is a concern.
Topics: Antipsychotic Agents; Drug Development; ERG1 Potassium Channel; Electrocardiography; Fluoroquinolones; Histamine Antagonists; Humans; Inhibitory Concentration 50; Long QT Syndrome; Torsades de Pointes
PubMed: 29380488
DOI: 10.1002/phar.2087 -
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 -
Cellular and Molecular Biology... Dec 2017Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether à-go-go-1 (Eag1) potassium channels...
Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether à-go-go-1 (Eag1) potassium channels display oncogenic properties, and have been suggested as early tumor markers and therapeutic targets for different cancers. These channels are overexpressed in many human tumors including PC. Astemizole targets several molecules involved in cancer including Eag1 channels, histamine receptors and ABC transporters. Here we studied Eag1 mRNA expression and protein levels in the non-tumorigenic and non-invasive human prostate RWPE-1 cell line, and in the tumorigenic and highly invasive human prostate WPE1-NB26 cell lines. The effect of astemizole on cell proliferation and apoptosis was also studied. The human prostate cell lines RWPE-1 and WPE1-NB26 were cultured following the provider´s instructions. Eag1 mRNA expression and protein levels were studied by real time RT-PCR and immunocytochemistry, respectively. Cell proliferation and apoptosis were studied by a fluorescence AlamarBlue® assay and flow cytometry, respectively. No difference in Eag1 mRNA expression was observed between the cell lines. However, high Eag1 protein levels were observed in the invasive WPE1-NB26 cells, in contrast to the weak protein expression in RWPE-1 cells. Accordingly, astemizole decreased cell proliferation at nanomolar concentrations only in the invasive WPE1-NB26 cells. Our results suggest that astemizole may have clinical relevance for prostate cancer treatment in patients with high Eag1 protein levels.
Topics: Apoptosis; Astemizole; Cell Line, Tumor; Cell Movement; Cell Proliferation; Ether-A-Go-Go Potassium Channels; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Prostatic Neoplasms; RNA, Messenger; Real-Time Polymerase Chain Reaction
PubMed: 29307346
DOI: 10.14715/cmb/2017.63.12.4 -
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