RESUMEN
Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human Cmax value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development.
Asunto(s)
Antineoplásicos/toxicidad , Cardiotoxinas/toxicidad , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Microscopía Electrónica/métodos , Miocitos Cardíacos/efectos de los fármacos , Cardiotoxicidad/patología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Colorantes Fluorescentes/análisis , Predicción , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/patologíaRESUMEN
ß1-adrenergic receptor (Adrb1) belongs to the superfamily of G-protein-coupled receptors (GPCRs) and plays a critical role in the regulation of heart rate and myocardial contraction force. GPCRs are phosphorylated at multiple sites to regulate distinct signal transduction pathways in different tissues. However, little is known about the location and function of distinct phosphorylation sites of Adrb1 in vivo. To clarify the mechanisms underlying functional regulation associated with Adrb1 phosphorylation in vivo, we aimed to identify Adrb1 phosphorylation sites in the mouse heart using phosphoproteomics techniques with nano-flow liquid chromatography/tandem mass spectrometry (LC-MS/MS). We revealed the phosphorylation residues of Adrb1 to be Ser274 and Ser280 in the third intracellular loop and Ser412, Ser417, Ser450, Ser451, and Ser462 at the C-terminus. We also found that phosphorylation at Ser274, Ser280, and Ser462 was enhanced in response to stimulation with an Adrb1 agonist. This is the first study to identify Adrb1 phosphorylation sites in vivo. These findings will provide novel insights into the regulatory mechanisms mediated by Adrb1 phosphorylation.
Asunto(s)
Miocardio/química , Miocardio/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/metabolismo , Animales , Cromatografía Liquida , Corazón , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteómica , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Mitochondrial fatty acid oxidation (mtFAO) plays an important role in hepatic energy metabolism. Severe mtFAO injury leads to nonalcoholic fatty liver disease (NAFLD) and liver failure. Several drugs have been withdrawn owing to safety issues, such as induction of fatty liver disease through mtFAO disruption. For instance, the antimicrobial triclocarban (TCC), an environmental contaminant that was removed from the market due to its unknown safety in humans, induces NAFLD in rats and promotes hepatic FAO in mice. Therefore, there are no consistent conclusions regarding the effects of TCC on FAO and lipid droplet accumulation. We hypothesized that TCC induces lipid droplet accumulation by inhibiting mtFAO in human hepatocytes. Here, we evaluated mitochondrial respiration in HepaRG cells to investigate the effects of TCC on fatty acid-driven oxidation in cells, electron transport chain parameters, lipid droplet accumulation, and antioxidant genes. The results suggest that TCC increases oxidative stress gene expression (GCLM, p62, HO-1, and NRF2) through lipid droplet accumulation via mtFAO inhibition in HepaRG cells. The results of the present study provide further insights into the effect of TCC on human NAFLD through mtFAO inhibition, and further in vivo studies could be used to validate the mechanisms.
Asunto(s)
Carbanilidas , Ácidos Grasos , Hepatocitos , Gotas Lipídicas , Oxidación-Reducción , Estrés Oxidativo , Humanos , Estrés Oxidativo/efectos de los fármacos , Carbanilidas/toxicidad , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Gotas Lipídicas/metabolismo , Gotas Lipídicas/efectos de los fármacos , Ácidos Grasos/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Línea Celular , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Metabolismo de los Lípidos/efectos de los fármacosRESUMEN
BACKGROUND: Sunitinib is known to cause cardiotoxicity in clinical settings. However, among sunitinib-treated patients experiencing adverse cardiac events, decreased cardiac function was reportedly reversible in > 50% of the patients. We previously showed that anti-cancer drugs such as sunitinib cause marked sarcomere disruption in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and the extent of sarcomere disruption can be used to predict drug-induced cardiotoxicity in humans. The aim of this study is to investigate whether the reversibility of sunitinib-induced cardiac events in clinical settings can be mimicked in vitro, and to examine the molecular mechanism responsible for sunitinib-induced cardiotoxicity focusing on the Hippo pathway. METHODS: iPSC-CMs were stimulated with sunitinib for 72 h and the morphology of sarcomere structures were analyzed by high-content analysis before and after sunitinib washout. To examine the involvement of the Hippo pathway in the sunitinib-induced sarcomere disruption, the extent of nuclear localization of YAP1 (yes-associated protein 1, a Hippo signaling target) was determined. iPSC-CMs were also stimulated with sunitinib and a small molecule inhibitor of the Hippo pathway, XMU-MP-1 and sarcomere structures were analyzed. RESULTS: We observed a spontaneous recovery in cardiac sarcomeres in iPSC-CMs that were significantly disrupted by sunitinib treatment after a 72 h or 144 h washout of sunitinib. The extent of nuclear localization of YAP1 was significantly reduced after sunitinib stimulation and tended to return to normal levels after drug washout. Simultaneous stimulation of iPSC-CM with sunitinib and XMU-MP-1 suppressed the sunitinib-induced disruption of sarcomeres. CONCLUSIONS: These results indicate that iPSC-CMs have the ability to recover from sunitinib-induced sarcomere disruption, and the Hippo pathway plays a role in the process of sunitinib-induced disruption of sarcomere and its recovery. Inhibition of the Hippo pathway may help to develop a co-medication strategy for mitigating the risk of sunitinib-induced adverse cardiac events.
Asunto(s)
Antineoplásicos/administración & dosificación , Vía de Señalización Hippo/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Sulfonamidas/administración & dosificación , Sunitinib/administración & dosificación , Células Cultivadas , Humanos , Miocitos Cardíacos/metabolismo , Sarcómeros/efectos de los fármacos , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Organoids are three-dimensional structures fabricated in vitro from pluripotent stem cells or adult tissue stem cells via a process of self-organization that results in the formation of organ-specific cell types. Human organoids are expected to mimic complex microenvironments and many of the in vivo physiological functions of relevant tissues, thus filling the translational gap between animals and humans and increasing our understanding of the mechanisms underlying disease and developmental processes. In the last decade, organoid research has attracted increasing attention in areas such as disease modeling, drug development, regenerative medicine, toxicology research, and personalized medicine. In particular, in the field of toxicology, where there are various traditional models, human organoids are expected to blaze a new path in future research by overcoming the current limitations, such as those related to differences in drug responses among species. Here, we discuss the potential usefulness, limitations, and future prospects of human liver, heart, kidney, gut, and brain organoids from the viewpoints of predictive toxicology research and drug development, providing cutting edge information on their fabrication methods and functional characteristics.
RESUMEN
Drug-induced toxicity remains one of the leading causes of discontinuation of the drug candidate and post-marketing withdrawal. Thus, early identification of the drug candidates with the potential for toxicity is crucial in the drug development process. With the recent discovery of human- Induced Pluripotent Stem Cells (iPSC) and the establishment of the differentiation protocol of human iPSC into the cell types of interest, the differentiated cells from human iPSC have garnered much attention because of their potential applicability in toxicity evaluation as well as drug screening, disease modeling and cell therapy. In this review, we expanded on current information regarding the feasibility of human iPSC-derived cells for the evaluation of drug-induced toxicity with a focus on human iPSCderived hepatocyte (iPSC-Hep), cardiomyocyte (iPSC-CMs) and neurons (iPSC-Neurons). Further, we CSAHi, Consortium for Safety Assessment using Human iPS Cells, reported our gene expression profiling data with DNA microarray using commercially available human iPSC-derived cells (iPSC-Hep, iPSC-CMs, iPSC-Neurons), their relevant human tissues and primary cultured human cells to discuss the future direction of the three types of human iPSC-derived cells.
Asunto(s)
Descubrimiento de Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/efectos de los fármacos , Neuronas/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Neuronas/citología , Neuronas/metabolismoRESUMEN
Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Canalículos Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Ciclosporina/farmacología , Expresión Génica , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Ratones , Ratones TransgénicosRESUMEN
INTRODUCTION: Drug-induced inotropic change is a risk factor in drug development; thus, de-risking is desired in the early stages of drug development. Unlike proarrhythmic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), few in vitro models were validated to predict cardiac contractility. Motion field imaging (MFI), a high-resolution block matching-based optical flow technique, was expected to possess high quantitative predictivity in the detection of contraction speed. We aimed to establish an in vitro model to assess drug-induced contractile changes using hiPSC-CMs and MFI. METHODS: MFI was designed to noninvasively characterize cardiomyocyte contractile behavior by analyzing light microscope video images, and maximum contraction speed (MCS) was used as the index of contractility. Using MFI, 9 inactive compounds, 10 negative inotropes, and 10 positive inotropes were tested. Two negative chronotropes, ZD7288 and ivabradine, were also tested. To determine the sensitivity and specificity of the assay, the minimum effective concentration of the MCS was compared with the human effective total therapeutic concentration for 28 compounds in clinical use. RESULTS: For 8 negative and 8 positive inotropes, the effects observed in in vivo and clinical studies were detected in MFI assay. MFI assay showed negative chronotropic changes without inotropic changes. MFI assay presented sufficient specificity (83%) and sensitivity (88%), and RNA-sequence analysis provided an insight into the relationship between occurrence of the false compounds and target gene expression. DISCUSSION: We demonstrated the utility of MFI assay using hiPSC-CMs to assess drug-induced contractile function. These results will facilitate the de-risking of compounds during early drug development.
Asunto(s)
Cardiotónicos/efectos adversos , Cardiotoxicidad/diagnóstico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Células Cultivadas , Expresión Génica/efectos de los fármacos , Humanos , Factores de Riesgo , Sensibilidad y Especificidad , Grabación en Video/métodosRESUMEN
Protein function is mainly modulated by dynamic reversible or irreversible post-translational modifications. Among them, the identification of protein phosphorylation sites and changes in phosphorylation levels in vivo are of considerable interest for a better understanding of the protein function. Thus, effective strategies for the quantitative determination of phosphorylation degrees for low abundant proteins, for which antibodies are not available, are required in order to evaluate the functional regulation of proteins attributed to phosphorylation. In this study, we used the heart ß1-adrenergic receptor (Adrb1) as a model protein and developed FLAG-Adrb1 knock-in mice, in which the FLAG tag was inserted at the N-terminus of Adrb1. The phosphorylation sites and levels of Adrb1 in the heart were elucidated by immuno-affinity purification followed by quantitative mass spectrometry analysis using ion intensity ratio of the phosphorylated peptide versus corresponding unphosphorylated peptide. The phosphorylation levels at Ser274 and Ser462 of Adrb1 were approximately 0.25 and 0.0023. This effective strategy should be useful for not only analyzing site-specific phosphorylation levels of target proteins, but also quantifying the expression levels of proteins of interest when appropriate antibodies are not available.
RESUMEN
Previously, we histochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovarian follicles, and demonstrated a marked reduction in the expression of TRAIL-decoy receptor-1 (DcRI) in granulosa cells of atretic follicles. In the present study, to confirm the inhibitory activity of DcR1 in granulosa cells, granulosa cells prepared from healthy follicles were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to cleave glycophospholipid anchor of DcR1 and to remove DcR1 from the cell surface, and then incubated with TRAIL. PI-PLC treatment increased the number of apoptotic cells induced by TRAIL. The present finding indicated the possibility that TRAIL and its receptors were involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.
Asunto(s)
Apoptosis , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Folículo Ovárico/citología , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Células Cultivadas , Femenino , Proteínas Ligadas a GPI , Células de la Granulosa/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/farmacología , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Porcinos , Ligando Inductor de Apoptosis Relacionado con TNF , Receptores Señuelo del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
To reveal the molecular regulation mechanism of selective follicular atresia in porcine ovaries, we isolated the porcine cDNA encoding cellular FLICE-like inhibitory protein (cFLIP), which inhibits death receptor-mediated apoptosis signal transduction. Two alternative splicing isoforms of cFLIP, porcine cellular FLIP-short form (pcFLIPS, 642 bp and 214-aa) and -long form (pcFLIPL, 1446 bp and 482-aa), were identified from a cDNA library prepared from follicular granulosa cells of pig ovaries. pcFLIPS and pcFLIPL indicated high identities with human and murine cFLIP, and both of them contain two tandem specific amino acid regions (death effector domain: DED) in their N-terminal, suggesting that pcFLIPS and pcFLIPL inhibit the death receptor-mediated apoptosis signal by binding to other pro-apoptotic factors mediated by DED. pcFLIPS contains a short C-terminal region, while pcFLIPL has a caspase-like domain in the C-terminal region. The reverse transcription-polymerase chain reaction analysis revealed that both pcFLIPS and pcFLIPL mRNAs were highly expressed in granulosa cells of healthy follicles, suggesting that these cFLIPs play important roles in the regulation mechanism of apoptosis in ovarian follicular granulosa cells. The present data will contribute to understanding of the physiological roles of cFLIPs in the apoptosis regulation in porcine tissues.
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Apoptosis/fisiología , Atresia Folicular/fisiología , Células de la Granulosa/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Clonación Molecular , Femenino , Células de la Granulosa/citología , Humanos , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Sus scrofaRESUMEN
To reveal the molecular mechanism of selective follicular atresia in porcine ovaries, we investigated the changes in the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (DR4) proteins and TRAIL mRNA in granulosa cells during follicular atresia. Immunohistochemical, Western immunoblotting and reverse transcription-polymerase chain reaction analyses (RT-PCR) revealed that significant increases in TRAIL protein and mRNA levels but not DR4 protein were changed during atresia. The RT-PCR product was confirmed to be porcine TRAIL by the cDNA sequence determination. An in vitro apoptosis inducing assay using cultured granulosa cells prepared from healthy follicles showed that TRAIL could activate caspase-3 and induce apoptotic cell death in the cells. The present findings confirm that TRAIL induces apoptosis in granulosa cells during atresia in porcine ovaries.
Asunto(s)
Apoptosis/fisiología , Atresia Folicular/fisiología , Células de la Granulosa/citología , Glicoproteínas de Membrana/fisiología , Ovario/citología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Caspasa 3 , Caspasas/metabolismo , ADN Complementario/genética , Activación Enzimática , Femenino , Atresia Folicular/genética , Expresión Génica , Células de la Granulosa/fisiología , Técnicas In Vitro , Glicoproteínas de Membrana/genética , Ovario/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/fisiología , Transducción de Señal , Sus scrofa , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Tumor necrosis factor (TNF) alpha can induce both cell death and cell proliferation and exerts its effects by binding to either TNF receptor (TNFR) 1 or 2. When TNFalpha-bound TNFR2 interacts with TNFR-associated factor 2 (TRAF2), expression of survival/antiapoptotic genes is up-regulated. In the present study we determined the changes in localization of TNFalpha and TRAF2 and their mRNAs and the expression of TNFR2 in granulosa cells during follicular atresia in pig ovaries. In healthy follicles, intense signals for TNFalpha and TRAF2 and their mRNAs were demonstrated in the outer zone of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased or trace staining for TRAF2 and its mRNA and decreased expression of TNFR2 were observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFalpha acts as a survival factor in granulosa cells during follicular atresia in pig ovaries.