RESUMEN
A large hexanucleotide (G4C2) repeat expansion in the first intronic region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several mechanisms have been proposed to explain how the repeat expansion drives disease, and we hypothesize that a variant-selective approach, in which transcripts affected by the repeat expansion are preferentially decreased, has the potential to address most of them. We report a stereopure antisense oligonucleotide, WVE-004, that executes this variant-selective mechanism of action. WVE-004 dose-dependently and selectively reduces repeat-containing transcripts in patient-derived motor neurons carrying a C9orf72-repeat expansion, as well as in the spinal cord and cortex of C9 BAC transgenic mice. In mice, selective transcript knockdown was accompanied by substantial decreases in dipeptide-repeat proteins, which are pathological biomarkers associated with the repeat expansion, and by preservation of healthy C9orf72 protein expression. These in vivo effects were durable, persisting for at least 6 months. These data support the advancement of WVE-004 as an investigational stereopure antisense oligonucleotide targeting C9orf72 for the treatment of C9orf72-associated ALS or FTD.
RESUMEN
Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.
In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.
Asunto(s)
Oligonucleótidos Antisentido , Animales , Células Cultivadas , Sistema Nervioso Central , Guanidina/química , Ratones , Neuronas/efectos de los fármacos , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Fosforotioatos , Ribonucleasa H/metabolismoRESUMEN
Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer's disease and other tauopathies.
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Encéfalo/metabolismo , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas tau/metabolismo , Animales , Humanos , Indazoles/química , Indazoles/metabolismo , Indazoles/farmacología , Ratones , Terapia Molecular Dirigida , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , RatasRESUMEN
Kinase inhibitors have transformed the treatment of many cancers and are showing the same promise for other indications including inflammatory diseases. This class of drugs is one of the most predominant in the pharmaceutical industry, but development and clinical utility is often limited by a broad spectrum of cardiovascular (CV) toxicities including QT prolongation and arrhythmia, left ventricular dysfunction, congestive heart failure, ischemia and myocardial infarction, and hypertension. In this review article, we provide a broad overview of the spectrum of CV events detected in clinical trials of kinase inhibitors and the known and proposed on- and off-target links between kinase inhibitor targets and these specific cardiotoxicities. We also examine the unique features of kinase inhibitors that have impeded complete mechanistic understanding of kinase inhibitor-associated cardiotoxicities and contributed to the disconnect between preclinical predictions and clinical findings. We then discuss various in vitro models currently in use that are amenable for high-throughput screening as well as lower throughput models that are valuable for mechanistic insight. These physiologically relevant models, together with newer "omic"-wide approaches will help to increase our understanding of the mechanisms underlying kinase inhibitor-associated cardiotoxicity and enable rational design of kinase inhibitors in the future.
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Cardiotoxicidad/etiología , Inhibidores de Proteínas Quinasas/efectos adversos , Animales , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-related label warnings or prescribing restrictions. While this liability is widely recognized, designing safer kinase inhibitors (KI) requires knowledge of the causative kinase(s). Efforts to unravel the kinases have encountered pharmacology with nearly prohibitive complexity. At therapeutically relevant concentrations, KIs show promiscuity distributed across the kinome. Here, to overcome this complexity, 65 KIs with known kinome-scale polypharmacology profiles were assessed for effects on cardiomyocyte (CM) beating. Changes in human iPSC-CM beat rate and amplitude were measured using label-free cellular impedance. Correlations between beat effects and kinase inhibition profiles were mined by computation analysis (Matthews Correlation Coefficient) to identify associated kinases. Thirty kinases met criteria of having (1) pharmacological inhibition correlated with CM beat changes, (2) expression in both human-induced pluripotent stem cell-derived cardiomyocytes and adult heart tissue, and (3) effects on CM beating following single gene knockdown. A subset of these 30 kinases were selected for mechanistic follow up. Examples of kinases regulating processes spanning the excitation-contraction cascade were identified, including calcium flux (RPS6KA3, IKBKE) and action potential duration (MAP4K2). Finally, a simple model was created to predict functional cardiotoxicity whereby inactivity at three sentinel kinases (RPS6KB1, FAK, STK35) showed exceptional accuracy in vitro and translated to clinical KI safety data. For drug discovery, identifying causative kinases and introducing a predictive model should transform the ability to design safer KI medicines. For cardiovascular biology, discovering kinases previously unrecognized as influencing cardiovascular biology should stimulate investigation of underappreciated signaling pathways.
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Corazón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/toxicidad , Calcio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/enzimología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Proteínas Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cardiovascular (CV) toxicity is a leading cause of drug attrition and withdrawal. Introducing in vitro assays with higher throughput should permit earlier CV hazard identification and enable medicinal chemists to design-out liabilities. Heretofore, development of in vitro CV assays has been limited by the challenge of replicating integrated cardiovascular physiology while achieving the throughput and consistency required for screening. These challenges appear to be met with a combination of human stem cell-derived cardiomyocytes (CM) which beat spontaneously and monitoring the response with technology that can assess drug-induced changes in voltage dependent contraction such as cellular impedance which has been validated with excellent predictivity for drug-induced arrhythmia and contractility. Here, we review advances in cardiomyocyte impedance with emphasis on stem cell-derived cardiomyocyte models for toxicity screening. Key perspectives include: the electrical principles of impedance technology, impedance detection of cardiomyocyte beating, beat parameter selection/analysis, validation in toxicity and drug discovery, and future directions. As a conclusion, an in vitro screening cascade is proffered using the downstream, inclusive detection of CM impedance assays as a primary screen followed by complementary CM assays chosen to enable mechanism-appropriate follow-up. The combined approach will enhance testing for CV liabilities prior to traditional in vivo models.
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Cardiotoxinas/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Cardiotoxicidad/diagnóstico , Cardiotoxicidad/patología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Impedancia Eléctrica , Humanos , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Células Madre/patología , Células Madre/fisiologíaRESUMEN
Cardiovascular toxicity, a prominent reason for late-stage failures in drug development, has resulted in a demand for in vitro assays that can predict this liability in early drug discovery. Current in vitro cardiovascular safety testing primarily focuses on ion channel modulation and low throughput cardiomyocyte (CM) contractility measurements. We evaluated both human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and rat neonatal CMs (rat CMs) on the xCELLigence Cardio system which uses impedance technology to quantify CM beating properties in a 96-well format. Forty-nine compounds were tested in concentration-response mode to determine potency for modulation of CM beating, a surrogate biomarker for contractility. These compounds had previously been tested in vivo and in a low throughput in vitro optical-based contractility assay that measures sarcomere shortening in electrically paced dog CMs. In comparison with in vivo contractility effects, hiPSC-CM impedance had assay sensitivity, specificity, and accuracy values of 90%, 74%, and 82%, respectively. These values compared favorably to values reported for the dog CM optical assay (83%, 84%, and 82%) and were slightly better than impedance using rat CMs (77%, 74%, and 74%). The potency values from the hiPSC-CM and rat CM assays spanned four orders of magnitude and correlated with values from the dog CM optical assay (r(2 )= 0.76 and 0.70, respectively). The Cardio system assay has >5× higher throughput than the optical assay. Thus, hiPSC-CM impedance testing can help detect the human cardiotoxic potential of novel therapeutics early in drug discovery, and if a hazard is identified, has sufficient throughput to support the design-make-test-analyze cycle to mitigate this liability.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Preparaciones Farmacéuticas/análisis , Animales , Animales Recién Nacidos , Cardiotoxicidad , Perros , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/fisiopatología , Impedancia Eléctrica , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Valor Predictivo de las Pruebas , Ratas , Sarcómeros/efectos de los fármacos , Sarcómeros/patologíaRESUMEN
Cardiovascular (CV) toxicity is a leading contributor to drug attrition. Implementing earlier testing has successfully reduced human Ether-à-go-go-Related Gene-related arrhythmias. How- ever, analogous assays targeting functional CV effects remain elusive. Demand to address this gap is particularly acute for kinase inhibitors (KIs) that suffer frequent CV toxicity. The drug class also presents some particularly challenging requirements for assessing functional CV toxicity. Specifically, an assay must sense a downstream response that integrates diverse kinase signaling pathways. In addition, sufficient throughput is essential for handling inherent KI nonselectivity. A new opportunity has emerged with cellular impedance technology, which detects spontaneous beating cardiomyocytes. Impedance assays sense morphology changes downstream of cardiomyocyte contraction. To evaluate cardiomyocyte impedance assays for KI screening, we investigated two distinct KI classes where CV toxicity was discovered late and target risks remain unresolved. Microtubule-associated protein/microtubule affinity regulating kinase (MARK) inhibitors decrease blood pressure in dogs, whereas checkpoint kinase (Chk) inhibitors (AZD7762, SCH900776) exhibit dose-limiting CV toxicities in clinical trials. These in vivo effects manifested in vitro as cardiomyocyte beat cessation. MARK effects were deemed mechanism associated because beat inhibition potencies correlated with kinase inhibition, and gene knockdown and microtubule-targeting agents suppressed beating. MARK inhibitor impedance and kinase potencies aligned with rat blood pressure effects. Chk inhibitor effects were judged off-target because Chk and beat inhibition potencies did not correlate and knockdowns did not alter beating. Taken together, the data demonstrate that cardiomyocyte impedance assays can address three unmet needs-detecting KI functional cardiotoxicity in vitro, determining mechanism of action, and supporting safety structure-activity relationships.
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Sistema Cardiovascular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Inhibidores de Proteínas Quinasas/farmacología , Animales , Perros , Masculino , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ratas , Ratas WistarRESUMEN
Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display 'UVA-mimetic' effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts.
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Catepsina B/efectos de la radiación , Catepsina L/efectos de la radiación , Fibroblastos/efectos de la radiación , Lisosomas/efectos de la radiación , Rayos Ultravioleta , Autofagia , Catepsina B/antagonistas & inhibidores , Catepsina L/antagonistas & inhibidores , Células Cultivadas , Fibroblastos/efectos de los fármacos , Humanos , Queratinocitos/enzimología , Queratinocitos/efectos de la radiación , Melanocitos/enzimología , Melanocitos/efectos de la radiación , Estrés Oxidativo , ARN Interferente Pequeño/farmacología , Piel/metabolismo , Piel/efectos de la radiaciónRESUMEN
D-Penicillamine (3,3-dimethyl-D-cysteine; DP) is an FDA-approved redox-active D-cysteine-derivative with antioxidant, disulfide-reducing, and metal chelating properties used therapeutically for the control of copper-related pathology in Wilson's disease and reductive cystine-solubilization in cystinuria. Based on the established sensitivity of metastatic melanoma cells to pharmacological modulation of cellular oxidative stress, we tested feasibility of using DP for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo. DP treatment induced caspase-dependent cell death in cultured human metastatic melanoma cells (A375, G361) without compromising viability of primary epidermal melanocytes, an effect not observed with the thiol-antioxidants N-acetyl-L-cysteine (NAC) and dithiothreitol. Focused gene expression array analysis followed by immunoblot detection revealed that DP rapidly activates the cytotoxic unfolded protein response (UPR; involving phospho-PERK, phospho-eIF2α, Grp78, CHOP, and Hsp70) and the mitochondrial pathway of apoptosis with p53 upregulation and modulation of Bcl-2 family members (involving Noxa, Mcl-1, and Bcl-2). DP (but not NAC) induced oxidative stress with early impairment of glutathione homeostasis and mitochondrial transmembrane potential. SiRNA-based antagonism of PMAIP1 expression blocked DP-induced upregulation of the proapoptotic BH3-only effector Noxa and prevented downregulation of the Noxa-antagonist Mcl-1, rescuing melanoma cells from DP-induced apoptosis. Intraperitoneal administration of DP displayed significant antimelanoma activity in a murine A375 xenograft model. It remains to be seen if melanoma cell-directed induction of UPR and apoptosis using DP or improved DP-derivatives can be harnessed for future chemotherapeutic intervention.
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Melanoma/tratamiento farmacológico , Penicilamina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/farmacología , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Humanos , Melanoma/patología , Ratones , Mitocondrias/efectos de los fármacos , Trasplante de Neoplasias , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Transcriptoma , Trasplante HeterólogoRESUMEN
Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B', HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma.
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Melanocitos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Tioestreptona/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Células Epidérmicas , Epidermis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico/efectos de los fármacos , Respuesta al Choque Térmico/genética , Hemo-Oxigenasa 1/genética , Humanos , Melanocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/genéticaRESUMEN
Recent research suggests that altered redox control of melanoma cell survival, proliferation, and invasiveness represents a chemical vulnerability that can be targeted by pharmacological modulation of cellular oxidative stress. The endoperoxide artemisinin and semisynthetic artemisinin-derivatives including dihydroartemisinin (DHA) constitute a major class of antimalarials that kill plasmodium parasites through induction of iron-dependent oxidative stress. Here, we demonstrate that DHA may serve as a redox chemotherapeutic that selectively induces melanoma cell apoptosis without compromising viability of primary human melanocytes. Cultured human metastatic melanoma cells (A375, G361, LOX) were sensitive to DHA-induced apoptosis with upregulation of cellular oxidative stress, phosphatidylserine externalization, and activational cleavage of procaspase 3. Expression array analysis revealed DHA-induced upregulation of oxidative and genotoxic stress response genes (GADD45A, GADD153, CDKN1A, PMAIP1, HMOX1, EGR1) in A375 cells. DHA exposure caused early upregulation of the BH3-only protein NOXA, a proapototic member of the Bcl2 family encoded by PMAIP1, and genetic antagonism (siRNA targeting PMAIP1) rescued melanoma cells from apoptosis indicating a causative role of NOXA-upregulation in DHA-induced melanoma cell death. Comet analysis revealed early DHA-induction of genotoxic stress accompanied by p53 activational phosphorylation (Ser 15). In primary human epidermal melanocytes, viability was not compromised by DHA, and oxidative stress, comet tail moment, and PMAIP1 (NOXA) expression remained unaltered. Taken together, these data demonstrate that metastatic melanoma cells display a specific vulnerability to DHA-induced NOXA-dependent apoptosis and suggest feasibility of future anti-melanoma intervention using artemisinin-derived clinical redox antimalarials.
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Antimaláricos/farmacología , Apoptosis/efectos de los fármacos , Artemisininas/farmacología , Melanocitos/efectos de los fármacos , Melanocitos/patología , Melanoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antioxidantes/farmacología , Apoptosis/genética , Caspasa 3/metabolismo , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/genética , Daño del ADN/genética , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Epidermis/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano/genética , Humanos , Quelantes del Hierro/farmacología , Melanocitos/metabolismo , Melanoma/genética , Melanoma/ultraestructura , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J cm(-2), twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and α-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, α-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage.
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Autofagia , Catepsina B/antagonistas & inhibidores , Fibroblastos/efectos de la radiación , Lisosomas/enzimología , Piel/efectos de la radiación , Rayos Ultravioleta , Electroforesis en Gel de Poliacrilamida , Fibroblastos/enzimología , Citometría de Flujo , Humanos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , Piel/citología , Piel/enzimologíaRESUMEN
Zinc ion homeostasis plays an important role in human cutaneous biology where it is involved in epidermal differentiation and barrier function, inflammatory and antimicrobial regulation, and wound healing. Zinc-based compounds designed for topical delivery therefore represent an important class of cutaneous therapeutics. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in over-the-counter topical antimicrobials, and has also been examined as an investigational therapeutic targeting psoriasis and UVB-induced epidermal hyperplasia. Recently, we have demonstrated that cultured primary human skin keratinocytes display an exquisite sensitivity to nanomolar ZnPT concentrations causing induction of heat shock response gene expression and poly(ADP-ribose) polymerase (PARP)-dependent cell death (Cell Stress Chaperones 15:309-322, 2010). Here we demonstrate that ZnPT causes rapid accumulation of intracellular zinc in primary keratinocytes as observed by quantitative fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS), and that PARP activation, energy crisis, and genomic impairment are all antagonized by zinc chelation. In epidermal reconstructs (EpiDerm™) exposed to topical ZnPT (0.1-2% in Vanicream™), ICP-MS demonstrated rapid zinc accumulation, and expression array analysis demonstrated upregulation of stress response genes encoding metallothionein-2A (MT2A), heat shock proteins (HSPA6, HSPA1A, HSPB5, HSPA1L, DNAJA1, HSPH1, HSPD1, HSPE1), antioxidants (SOD2, GSTM3, HMOX1), and the cell cycle inhibitor p21 (CDKN1A). IHC analysis of ZnPT-treated EpiDerm™ confirmed upregulation of Hsp70 and TUNEL-positivity. Taken together our data demonstrate that ZnPT impairs zinc ion homeostasis and upregulates stress response gene expression in primary keratinocytes and reconstructed human epidermis, activities that may underlie therapeutic and toxicological effects of this topical drug.
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Epidermis/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Homeostasis/efectos de los fármacos , Compuestos Organometálicos/farmacología , Estrés Oxidativo/efectos de los fármacos , Piridinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Zinc/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epidérmicas , Epidermis/metabolismo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Estrés Oxidativo/genética , Relación Estructura-ActividadRESUMEN
Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B', p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.
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2,6-Dicloroindofenol/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Estrés Oxidativo , 2,6-Dicloroindofenol/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/genética , Caspasas/metabolismo , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Daño del ADN , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Glutatión/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Immunoblotting , Potencial de la Membrana Mitocondrial , Ratones , NAD(P)H Deshidrogenasa (Quinona)/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , ARN Interferente PequeñoRESUMEN
Solar UVA exposure plays a causative role in skin photoaging and photocarcinogenesis. Here, we describe the proteomic identification of novel UVA-targets in human dermal fibroblasts following a two-dimensional-difference-gel-electrophoresis (2D-DIGE) approach. Fibroblasts were exposed to noncytotoxic doses of UVA or left untreated, and total protein extracts underwent CyDye-labeling followed by 2D-DIGE/mass-spectrometric identification of differentially expressed proteins, confirmed independently by immunodetection. The protein displaying the most pronounced UVA-induced upregulation was identified as the nucleolar protein nucleophosmin. The protein undergoing the most pronounced UVA-induced downregulation was identified as cathepsin B, a lysosomal cysteine-protease displaying loss of enzymatic activity and altered maturation after cellular UVA exposure. Extensive lysosomal accumulation of lipofuscin-like autofluorescence and osmiophilic material occurred in UVA-exposed fibroblasts as detected by confocal fluorescence microscopy and transmission electron microscopy, respectively. Array analysis indicated UVA-induced upregulation of oxidative stress response gene expression, and UVA-induced loss of cathepsin B enzymatic activity in fibroblasts was suppressed by antioxidant intervention. Pharmacological cathepsin B inhibition using CA074Me mimicked UVA-induced accumulation of lysosomal autofluorescence and deficient cathepsin B maturation. Taken together, these data support the hypothesis that cathepsin B is a crucial target of UVA-induced photo-oxidative stress causatively involved in dermal photodamage through the impairment of lysosomal removal of lipofuscin.
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Catepsina B/metabolismo , Proteínas Nucleares/metabolismo , Piel/metabolismo , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Acetilcisteína/farmacología , Antioxidantes/farmacología , Línea Celular , Dipéptidos/farmacología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Perfilación de la Expresión Génica , Humanos , Lipofuscina/metabolismo , Lisosomas/metabolismo , Nucleofosmina , Estrés Oxidativo , Proteómica , Envejecimiento de la Piel/genética , Envejecimiento de la Piel/fisiologíaRESUMEN
Light-driven electron and energy transfer involving non-DNA skin chromophores as endogenous photosensitizers induces oxidative stress in UVA-exposed human skin, a process relevant to photoaging and photocarcinogenesis. Malondialdehyde is an electrophilic dicarbonyl-species derived from membrane lipid peroxidation. Here, we present experimental evidence suggesting that the malondialdehyde-derived protein epitope dihydropyridine (DHP)-lysine is a potent endogenous UVA-photosensitizer of human skin cells. Immunohistochemical analysis revealed the abundant occurrence of malondialdehyde-derived and DHP-lysine epitopes in human skin. Using the chemically protected dihydropyridine-derivative (2S)-Boc-2-amino-6-(3,5-diformyl-4-methyl-4H-pyridin-1-yl)-hexanoic acid-t-butylester as a model of peptide-bound DHP-lysine, photodynamic inhibition of proliferation and induction of cell death were observed in human skin Hs27 fibroblasts as well as primary and HaCaT keratinocytes exposed to the combined action of UVA and DHP-lysine. DHP-lysine photosensitization induced intracellular oxidative stress, p38 MAPkinase activation, and upregulation of heme oxygenase-1 expression. Consistent with UVA-driven ROS formation from DHP-lysine, formation of superoxide, hydrogen peroxide, and singlet oxygen was detected in chemical assays, but little protection was achieved using SOD or catalase during cellular photosensitization. In contrast, inclusion of NaN(3) completely abolished DHP-photosensitization. Taken together, these data demonstrate photodynamic activity of DHP-lysine and support the hypothesis that malondialdehyde-derived protein-epitopes may function as endogenous sensitizers of UVA-induced oxidative stress in human skin.
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Lisina/análogos & derivados , Malondialdehído/química , Estrés Oxidativo , Fármacos Fotosensibilizantes/química , Piridinas/química , Piel/efectos de la radiación , Rayos Ultravioleta , Apoptosis , Catalasa/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Hemo-Oxigenasa 1/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Lisina/química , Especies Reactivas de Oxígeno/metabolismo , Piel/efectos de los fármacos , Azida Sódica/farmacología , Superóxido Dismutasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Colorectal cancer (CRC) is a major cause of tumor-related morbidity and mortality worldwide. Recent research suggests that pharmacological intervention using dietary factors that activate the redox sensitive Nrf2/Keap1-ARE signaling pathway may represent a promising strategy for chemoprevention of human cancer including CRC. In our search for dietary Nrf2 activators with potential chemopreventive activity targeting CRC, we have focused our studies on trans-cinnamic aldehyde (cinnamaldeyde, CA), the key flavor compound in cinnamon essential oil. Here we demonstrate that CA and an ethanolic extract (CE) prepared from Cinnamomum cassia bark, standardized for CA content by GC-MS analysis, display equipotent activity as inducers of Nrf2 transcriptional activity. In human colon cancer cells (HCT116, HT29) and non-immortalized primary fetal colon cells (FHC), CA- and CE-treatment upregulated cellular protein levels of Nrf2 and established Nrf2 targets involved in the antioxidant response including heme oxygenase 1 (HO-1) and gamma-glutamyl-cysteine synthetase (gamma-GCS, catalytic subunit). CA- and CE-pretreatment strongly upregulated cellular glutathione levels and protected HCT116 cells against hydrogen peroxide-induced genotoxicity and arsenic-induced oxidative insult. Taken together our data demonstrate that the cinnamon-derived food factor CA is a potent activator of the Nrf2-orchestrated antioxidant response in cultured human epithelial colon cells. CA may therefore represent an underappreciated chemopreventive dietary factor targeting colorectal carcinogenesis.
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Acroleína/análogos & derivados , Antioxidantes , Cinnamomum zeylanicum , Neoplasias del Colon/prevención & control , Factor 2 Relacionado con NF-E2/metabolismo , Acroleína/farmacología , Línea Celular Tumoral , Quimioprevención/métodos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Suplementos Dietéticos , Células Epiteliales , Humanos , Oxidación-Reducción , Transducción de SeñalRESUMEN
The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the aromatic hydrocarbon receptor (AhR), which contributes to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen receptor- alpha (ER alpha )-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. We report that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17 beta -estradiol (E2)-dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ER alpha , acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists.
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Anticarcinógenos/farmacología , Proteína BRCA1/metabolismo , Epigénesis Genética/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Estilbenos/farmacología , Proteína BRCA1/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Dibenzodioxinas Policloradas , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/genética , Resveratrol , Estilbenos/administración & dosificación , Transcripción GenéticaRESUMEN
Extracellular cytokine function of the non-histone nuclear protein high-mobility group box 1 (HMGB1) has recently been recognized as an important drug target for novel anti-inflammatory therapeutics. Accumulating evidence supports the mechanistic involvement of the alarmin HMGB1 in skin response to microbial infection and ultraviolet-induced solar damage. Moreover, HMGB1 modulation of inflammatory signaling and tissue remodeling is now emerging as a causative factor in wound repair, autoimmune dysregulation, and skin carcinogenesis, representing cutaneous pathologies that affect large patient populations with unmet therapeutic needs. Recent structure-based drug discovery efforts have aimed at increasing the number of small molecule- and biologics-based prototype therapeutics targeting HMGB1. Small molecule drugs that may provide therapeutic benefit through HMGB1-directed mechanisms involve HMGB1 inhibitory ligands, Toll-like receptor antagonists, RAGE antagonists, alpha7 nicotinic acetylcholine receptor agonists, G2A antagonists, serine protease inhibitors, and alpha-dicarbonyl-based soft electrophiles. Using some of these agents, pharmacological modulation of HMGB1-associated cutaneous pathology has been achieved with an acceptable toxicity profile, and preclinical proof-of-concept experimentation has demonstrated feasibility of developing HMGB1-modulators into novel systemic and topical therapeutics that target cutaneous inflammatory dysregulation.