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1.
J Toxicol Sci ; 46(8): 359-369, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34334557

RESUMO

Predicting drug-induced side effects in the cardiovascular system is very important because it can lead to the discontinuation of new drugs/candidates or the withdrawal of marketed drugs. Although chronic assessment of cardiac contractility is an important issue in safety pharmacology, an in vitro evaluation system has not been fully developed. We previously developed an imaging-based contractility assay system to detect acute cardiotoxicity using human iPS cell-derived cardiomyocytes (hiPSC-CMs). To extend the system to chronic toxicity assessment, we examined the effects of the anti-hepatitis C virus (HCV) drug candidate BMS-986094, a guanosine nucleotide analogue, which was withdrawn from phase 2 clinical trials because of unexpected contractility toxicities. Additionally, we examined sofosbuvir, another nucleotide analogue inhibitor of HCV that has been approved as an anti-HCV drug. Motion imaging analysis revealed the difference in cardiotoxicity between the cardiotoxic BMS-986094 and the less toxic sofosbuvir in hiPSC-CMs, with a minimum of 4 days of treatment. In addition, we found that BMS-986094-induced contractility impairment was mediated by a decrease in calcium transient. These data suggest that chronic treatment improves the predictive power for the cardiotoxicity of anti-HCV drugs. Thus, hiPSC-CMs can be a useful tool to assess drug-induced chronic cardiotoxicity in non-clinical settings.

2.
Nihon Yakurigaku Zasshi ; 156(4): 208-213, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-34193697

RESUMO

The mission of regulatory science is to promote human longevity by providing safer and more effective drugs and ensuring human health. At present, various in vitro and in vivo evaluation methods are used for drug development, and no major problems have been observed. However, there is still room for improvement in terms of risk prediction in humans. Thus, new approaches and methodologies (NAMs) have recently been developed to predict adverse events in humans more accurately. Based on the animal alternative methods and the current COVID-19 pandemic, in vitro methods, such as human iPS cells, and computational approach are accelerated to improve the efficiency of drug development, ensure the patients' safety and speed up the review process. In this review, we would like to summarize the current status and future perspectives of pharmacological assay system using NAM in drug development.


Assuntos
COVID-19 , Pandemias , Alternativas aos Testes com Animais , Animais , Desenvolvimento de Medicamentos , Humanos , SARS-CoV-2
3.
Methods Mol Biol ; 2320: 151-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302656

RESUMO

Human-induced pluripotent stem cell (iPSC) technology paves the way for next-generation drug-safety assessment. In particular, human iPSC-derived cardiomyocytes, which exhibit electrical activity, are useful as a human cell model for assessing QT-interval prolongation and the risk of the lethal arrhythmia Torsade de Pointes (TdP). In addition to proarrhythmia assay, contractile behavior has received increased attention in drug development. In this study, we developed a novel high-throughput in vitro assay system using motion vectors to evaluate the contractile activity of iPSC-derived cardiomyocytes as a physiologically relevant human platform. The methods presented here highlight the use of commercially available iPSC-derived cardiomyocytes, iCell cardiomyocytes, for contractility evaluation recorded by the motion vector system.


Assuntos
Bioensaio/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Arritmias Cardíacas/terapia , Células Cultivadas , Humanos , Síndrome do QT Longo/terapia , Torsades de Pointes/terapia
4.
Toxicol Sci ; 183(1): 227-239, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34142159

RESUMO

Coronavirus disease 2019 (COVID-19) continues to spread across the globe, with numerous clinical trials underway seeking to develop and test effective COVID-19 therapies, including remdesivir. Several ongoing studies have reported hydroxychloroquine-induced cardiotoxicity, including development of torsade de pointes (TdP). Meanwhile, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to serve as a tool for assessing drug-induced cardiotoxicity, such as TdP and contraction impairment. However, the cardiotoxicity of COVID-19 treatments has not been fully assessed using hiPSC-CMs. In this study, we focused on drug repurposing with various modes of actions and examined the TdP risk associated with COVID-19 treatments using field potential using multi-electrode array system and motion analysis with hiPSC-CMs. Hydroxychloroquine induced early after depolarization, while remdesivir, favipiravir, camostat, and ivermectin had little effect on field potentials. We then analyzed electromechanical window, which is defined as the difference between field potential and contraction-relaxation durations. Hydroxychloroquine decreased electromechanical window of hiPSC-CMs in a concentration-dependent manner. In contrast, other drugs had little effect. Our data suggest that hydroxychloroquine has proarrhythmic risk and other drugs have low proarrhythmic risk. Thus, hiPSC-CMs represent a useful tool for assessing the comprehensive cardiotoxicity caused by COVID-19 treatments in nonclinical settings.


Assuntos
COVID-19 , Células-Tronco Pluripotentes Induzidas , COVID-19/tratamento farmacológico , Cardiotoxicidade , Células Cultivadas , Humanos , Miócitos Cardíacos , SARS-CoV-2
5.
Front Cell Dev Biol ; 8: 542562, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33015053

RESUMO

Contractility of the human heart increases as its beating rate is elevated, so-called positive force-frequency relationship; however, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been reported to exert a negative force-frequency relationship. We tested the hypothesis that the regulation of motion directions by electrical pacing and/or oxygen supply may improve the electro-mechanical properties of hiPSC-CMs monolayers. To better evaluate the spatial and temporal relationship between electrical excitation and contractile motion, we simultaneously observed the field potential and motion vector of hiPSC-CMs sheets. Under spontaneous contraction, although an electrical excitation originating from a region propagated unidirectionally over the cell sheet, contraction wave started from multiple sites, and relaxation wave was initiated from a geometric center of hiPSC-CMs sheet. During electrical pacing, contraction and relaxation waves were propagated from the stimulated site. Interestingly, the maximum contraction speed was more increased when the hiPSC-CMs sheet was stimulated at an area relaxation initiated under spontaneous condition. Furthermore, motion vector analysis demonstrated that "positive contraction velocity-frequency relationship" in contraction and "frequency-dependent enhancement of relaxation" were produced in the cell sheet by optimizing the direction and order of the contractile motion with pacing at the relaxation-initiating area. A close analysis of motion vectors along with field potential recording demonstrated that relaxation process consists of fast and slow phases, and suggest that intracellular Ca2+ dynamics may be closely related to functions of Ca2+-ATPase pump and Na+-Ca2+ exchangers. Namely, the slow relaxation phase occurred after the second peak of field potential, suggesting that the slow phase may be associated with extrusion of Ca2+ by Na+-Ca2+ exchangers during repolarization. Increase of oxygen concentration from 20 to 95% as well as ß-adrenergic stimulation with isoproterenol accelerated the fast relaxation, suggesting that it could depend on Ca2+ uptake via Ca2+-ATPase during the depolarization phase. The ratio of maximum contraction speed to field potential duration was increased by the ß-adrenergic stimulation, indicating the elevated contraction efficiency per Ca2+-influx. Thus, these findings revealed potential ability of conventional monolayers of hiPSC-CMs, which will help apply them to translational study filling the gap between physiological as well as pharmacological studies and clinical practice.

6.
Nihon Yakurigaku Zasshi ; 155(3): 171-174, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32378638

RESUMO

Cardiac safety assessments play a key role in drug development. New non-clinical cardiac safety risk assessments, such as the use of iPSC-derived cardiomyocytes, have been validated by several consortiums both in Japan and abroad. The emerging multidisciplinary field of cardio-oncology has been recognized more important. The success of new cancer therapies has improved life expectancy of cancer patients, hence more attention has been paid to cardiotoxicities associated with existing and new anti-cancer therapies, such as cardiomyocyte injury and heart failure, vascular injury and hypertension or thrombosis, which accelerated coronary artery disease. In addition to the well-studied proarrhythmia risk, some cardiotoxicities, such as contractility impairment, are expected to be evaluated by iPSC-derived cardiomyocytes. Here we developed a novel imaging-based in vitro contractility assay using iPSC-derived cardiomyocytes. In the review, we would like to discuss the current status and future perspectives in the assessment of cardiac contractile function by anti-cancer agents.


Assuntos
Cardiologia/tendências , Células-Tronco Pluripotentes Induzidas/citologia , Oncologia/tendências , Miócitos Cardíacos/citologia , Cardiotoxicidade , Humanos , Pesquisa Interdisciplinar , Neoplasias/tratamento farmacológico
7.
Curr Pharm Biotechnol ; 21(9): 765-772, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31264543

RESUMO

Growing evidence suggests that Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) can be used as a new human cell-based platform to assess cardiac toxicity/safety during drug development. Cardiotoxicity assessment is highly challenging due to species differences and various toxicities, such as electrophysiological and contractile toxicities, which can result in proarrhythmia and heart failure. To explore proarrhythmic risk, the Multi-Electrode Array (MEA) platform is widely used to assess QT-interval prolongation and the proarrhythmic potential of drug candidates using hiPSC-CMs. Several consortiums, including the Comprehensive in vitro Proarrhythmia Assay (CiPA) and the Japanese iPS Cardiac Safety Assessment (JiCSA), have demonstrated the applicability of hiPSC-CMs/MEA for assessing the torsadogenic potential of drug candidates. Additionally, contractility is a key safety issue in drug development, and efforts have been undertaken to measure contractility by a variety of imaging-based methods using iPS-CMs. Therefore, hiPSC-CMs might represent a standard testing tool for evaluating the proarrhythmic and contractile potentials. This review provides new insights into the practical application of hiPSC-CMs in early or late-stage nonclinical testing during drug development.


Assuntos
Arritmias Cardíacas/induzido quimicamente , Desenvolvimento de Medicamentos , Drogas em Investigação/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Cardiotoxicidade , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Neoplasias , Medição de Risco
8.
PLoS Pathog ; 11(4): e1004763, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25875106

RESUMO

Human papillomaviruses infect stratified epithelia and link their productive life cycle to the differentiation state of the host cell. Productive viral replication or amplification is restricted to highly differentiated suprabasal cells and is dependent on the activation of the ATM DNA damage pathway. The ATM pathway has three arms that can act independently of one another. One arm is centered on p53, another on CHK2 and a third on SMC1/NBS1 proteins. A role for CHK2 in HPV genome amplification has been demonstrated but it was unclear what other factors provided important activities. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role together with NBS1 in the ATM DNA damage response. In normal cells, SMC1 becomes phosphorylated in response to radiation, however, in HPV positive cells our studies demonstrate that it is constitutively activated. Furthermore, pSMC1 is found localized in distinct nuclear foci in complexes with γ-H2AX, and CHK2 and bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 forms complexes with the insulator transcription factor CTCF and our studies show that these factors bind to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 open reading frame inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF to facilitate genome amplification. Our findings identify both SMC1 and CTCF as critical regulators of the differentiation-dependent life cycle of high-risk human papillomaviruses.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Queratinócitos/virologia , Papillomaviridae/fisiologia , Infecções por Papillomavirus/metabolismo , Proteínas Repressoras/metabolismo , Replicação Viral/fisiologia , Fator de Ligação a CCCTC , Diferenciação Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Imunofluorescência , Humanos , Immunoblotting , Queratinócitos/metabolismo , Mutagênese Sítio-Dirigida , Transfecção , Ativação Viral
9.
J Virol ; 89(3): 1919-24, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25410865

RESUMO

Amplification of human papillomaviruses (HPV) is dependent on the ATM DNA damage pathway. In cells with impaired p53 activity, DNA damage repair requires the activation of p38MAPK along with MAPKAP kinase 2 (MK2). In HPV-positive cells, phosphorylation of p38 and MK2 proteins was induced along with relocalization to the cytoplasm. Treatment with MK2 or p38 inhibitors blocked HPV genome amplification, identifying the p38/MK2 pathway as a key regulator of the HPV life cycle.


Assuntos
Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Papillomaviridae/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Replicação Viral , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular , Humanos , Fosforilação , Transporte Proteico
10.
Front Microbiol ; 4: 251, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23986755

RESUMO

Papillomavirus is the etiological agent for warts and several squamous carcinomas. Skin cancer induced by cottontail rabbit papillomavirus was the first animal model for virus-induced carcinogenesis. The target organ of the virus infection is stratified epithelium and virus replication is tightly regulated by the differentiation program of the host cell. E1^E4 protein is a viral gene product, and although it is considered to be involved in the control of virus replication, little is known about the biological role. We found that HPV18 E1^E4 was assembled into an aggresome-like compartment and was involved in sequestration of virus oncoproteins, which might contribute to the differentiation-dependent lifecycle of papillomavirus.

11.
Front Microbiol ; 3: 152, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22536200

RESUMO

Human papillomaviruses (HPVs) target the stratified epidermis, and can causes diseases ranging from benign condylomas to malignant tumors. Infections of HPVs in the genital tract are among the most common sexually transmitted diseases, and a major risk factor for cervical cancer. The virus targets epithelial cells in the basal layer of the epithelium, while progeny virions egress from terminally differentiated cells in the cornified layer, the surface layer of the epithelium. In infected basal cells, the virus maintains its genomic DNA at low-copy numbers, at which the viral productive lifecycle cannot proceed. Progression of the productive lifecycle requires differentiation of the host cell, indicating that there is tight crosstalk between viral replication and host differentiation programs. In this review, we discuss the regulation of the HPV lifecycle controlled by the differentiation program of the host cells.

12.
Mech Dev ; 128(11-12): 577-90, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22265871

RESUMO

Wnt signaling plays crucial roles in neural development. We previously identified Neucrin, a neural-specific secreted antagonist of canonical Wnt/ß-catenin signaling, in humans and mice. Neucrin has one cysteine-rich domain, in which the positions of 10 cysteine residues are similar to those in the second cysteine-rich domain of Dickkopfs, secreted Wnt antagonists. Here, we have identified zebrafish neucrin to understand its roles in vivo. Zebrafish Neucrin also has one cysteine-rich domain, which is significantly similar to that of mouse Neucrin. Zebrafish neucrin was also predominantly expressed in developing neural tissues. To examine roles of neucrin in neural development, we analyzed neucrin knockdown embryos. Neural development in zebrafish embryos was impaired by the knockdown of neucrin. The knockdown of neucrin caused increased expression of the Wnt/ß-catenin target genes. In contrast, overexpression of neucrin reduced the expression of the Wnt/ß-catenin target genes. The knockdown of neucrin affected specification of dorsal region in the midbrain and hindbrain. The knockdown of neucrin also suppressed neuronal differentiation and caused increased cell proliferation and apoptosis in developing neural tissues. Neucrin is a unique secreted Wnt antagonist that is predominantly expressed in developing neural tissues and plays roles in neural development in zebrafish.


Assuntos
Proteínas do Tecido Nervoso/fisiologia , Neurogênese , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Apoptose , Proteína Axina/genética , Proteína Axina/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Proliferação de Células , Sobrevivência Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
Cancer Sci ; 101(2): 536-42, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19917059

RESUMO

Human papillomaviruses (HPVs) infect the stratified epithelial organ. The infection induces benign tumors, which occasionally progress into malignant tumors. To elucidate the virus-induced tumorigenesis, an understanding of the lifecycle of HPV is crucial. In this report, we developed a new system for the analysis of the HPV lifecycle. The new system consists of a novel HPV replicon and an organotypic "raft" culture, by which the HPV-DNA is maintained stably in normal human keratinocytes for a long period and the viral vegetative replication is reproduced. This system will benefit biochemical and genetic studies on the lifecycle of HPV and tumorigenesis. This system is also valuable in screening for antiviral compounds. We confirmed its usefulness by evaluating the antivirus effect of cytokines.


Assuntos
Antivirais/farmacologia , Citocinas/farmacologia , Papillomavirus Humano 18/efeitos dos fármacos , Replicon/efeitos dos fármacos , Células Cultivadas , DNA Viral/análise , Papillomavirus Humano 18/genética , Humanos , Replicação Viral/efeitos dos fármacos
14.
J Virol ; 82(17): 8820-7, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18579583

RESUMO

Infection by human papillomavirus (HPV) is a major risk factor for human cervical carcinoma. However, the HPV infection alone is not sufficient for cancer formation. Cervical carcinogenesis is considered a multistep process accompanied by genetic alterations of the cell. Ras is activated in approximately 20% of human cancers, and it is related to the metastatic conversion of tumor cells. We investigated how Ras activation was involved in the malignant conversion of HPV-infected lesions. The active form of H-ras was introduced into human primary keratinocytes expressing the HPV type 18 (HPV18) oncoproteins E6 and/or E7. We analyzed the keratinocytes' growth potentials and found that the activation of the Ras pathway induced senescence-like growth arrest. Senescence could be eliminated by high-risk E7 expression, suggesting that the pRb pathway was important for Ras-induced senescence. Then we analyzed the effect of Ras activation on epidermis development by using an organotypic "raft" culture and found that the E7 and H-ras coexpressions conferred invasive potential on the epidermis. This invasiveness resulted from the upregulation of MT1-MMP and MMP9 by H-ras and E7, respectively, in which the activation of the MEK/extracellular signal-regulated kinase pathway was involved. These results indicated that the activation of Ras or the related signal pathways promoted the malignant conversion of HPV-infected cells.


Assuntos
Transformação Celular Neoplásica , Queratinócitos/fisiologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas ras/fisiologia , Técnicas de Cultura de Células , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Senescência Celular , Meios de Cultura Livres de Soro , Feminino , Vetores Genéticos , Células HeLa , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiologia , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Queratinócitos/virologia , Rim/citologia , Masculino , Mutação , Proteínas Oncogênicas Virais/metabolismo , Técnicas de Cultura de Órgãos , Papillomaviridae/fisiologia , Proteína do Retinoblastoma/metabolismo , Retroviridae/genética , Neoplasias do Colo do Útero/patologia
15.
Uirusu ; 58(2): 165-72, 2008 Dec.
Artigo em Japonês | MEDLINE | ID: mdl-19374194

RESUMO

Papillomavirus is a pathogenic virus that induces benign tumor at the infected lesion, and its association with malignant tumor was first identified by R. Shope using animal model. A variety of cancers have been reported to be associated with the infection of human papillomavirus since the report by H. zur Hausen that describes a connection between the HPV infection and cervical cancer. The HPV infection is broadly distributed as a sexually transmitted disease (STD) and recently the initial age diagnosed as the cervical cancer is getting lowered. Because of its clinical importance, the study on HPV has been focused on the oncogenic properties, and the results of which had great impacts on the researches of the tumor suppressors, such as p53 and pRb, and "ubiquiitn-proteasome" pathway. On the other hand, the biological properties of HPV remain mostly disclosed. The lifecycle of HPV is tightly linked to the differentiation program of the target epithelial cell, and this unique property has hampered the study on the HPV replication mechanism. Here we summarized the findings on the HPV lifecycle, including the virus gene functions, the regulation of viral gene expression and replication.


Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Papillomavirus Humano 16/fisiologia , Animais , Regulação Viral da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Genoma Viral , Papillomavirus Humano 16/genética , Humanos , Fases de Leitura Aberta/genética , Replicação Viral/genética
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