RESUMEN
Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias Glandulares y Epiteliales , Anticuerpos Biespecíficos/farmacología , Receptores ErbB/metabolismo , Humanos , Imidazoles , Neoplasias Glandulares y Epiteliales/metabolismo , Células Madre Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Doxorubicin is widely used in the treatment of different cancers, and its side effects can be severe in many tissues, including the intestines. Symptoms such as diarrhoea and abdominal pain caused by intestinal inflammation lead to the interruption of chemotherapy. Nevertheless, the molecular mechanisms associated with doxorubicin intestinal toxicity have been poorly explored. This study aims to investigate such mechanisms by exposing 3D small intestine and colon organoids to doxorubicin and to evaluate transcriptomic responses in relation to viability and apoptosis as physiological endpoints. The in vitro concentrations and dosing regimens of doxorubicin were selected based on physiologically based pharmacokinetic model simulations of treatment regimens recommended for cancer patients. Cytotoxicity and cell morphology were evaluated as well as gene expression and biological pathways affected by doxorubicin. In both types of organoids, cell cycle, the p53 signalling pathway, and oxidative stress were the most affected pathways. However, significant differences between colon and SI organoids were evident, particularly in essential metabolic pathways. Short time-series expression miner was used to further explore temporal changes in gene profiles, which identified distinct tissue responses. Finally, in silico proteomics revealed important proteins involved in doxorubicin metabolism and cellular processes that were in line with the transcriptomic responses, including cell cycle and senescence, transport of molecules, and mitochondria impairment. This study provides new insight into doxorubicin-induced effects on the gene expression levels in the intestines. Currently, we are exploring the potential use of these data in establishing quantitative systems toxicology models for the prediction of drug-induced gastrointestinal toxicity.
Asunto(s)
Doxorrubicina/toxicidad , Intestinos/efectos de los fármacos , Intestinos/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Colon/efectos de los fármacos , Doxorrubicina/farmacología , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Intestino Delgado/efectos de los fármacos , Modelos Biológicos , Organoides/citología , Organoides/efectos de los fármacos , Organoides/metabolismo , Proteómica , Transcriptoma/genéticaRESUMEN
Gefitinib is a tyrosine kinase inhibitor (TKI) that selectively inhibits the epidermal growth factor receptor (EGFR), hampering cell growth and proliferation. Due to its action, gefitinib has been used in the treatment of cancers that present abnormally increased expression of EGFR. However, side effects from gefitinib therapy may occur, among which diarrhoea is most common, that can lead to interruption of the planned therapy in the more severe cases. The mechanisms underlying intestinal toxicity induced by gefitinib are not well understood. Therefore, this study aims at providing insight into these mechanisms based on transcriptomic responses induced in vitro. A 3D culture of healthy human colon and small intestine (SI) organoids was exposed to 0.1, 1, 10 and 30 µM of gefitinib, for a maximum of three days. These drug concentrations were selected using physiologically-based pharmacokinetic simulation considering patient dosing regimens. Samples were used for the analysis of viability and caspase 3/7 activation, image-based analysis of structural changes, as well as RNA isolation and sequencing via high-throughput techniques. Differential gene expression analysis showed that gefitinib perturbed signal transduction pathways, apoptosis, cell cycle, FOXO-mediated transcription, p53 signalling pathway, and metabolic pathways. Remarkably, opposite expression patterns of genes associated with metabolism of lipids and cholesterol biosynthesis were observed in colon versus SI organoids in response to gefitinib. These differences in the organoids' responses could be linked to increased activated protein kinase (AMPK) activity in colon, which can influence the sensitivity of the colon to the drug. Therefore, this study sheds light on how gefitinib induces toxicity in intestinal organoids and provides an avenue towards the development of a potential tool for drug screening and development.
Asunto(s)
Gefitinib/farmacología , Intestinos/efectos de los fármacos , Organoides/efectos de los fármacos , Transcriptoma/genética , Anciano , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores ErbB/metabolismo , Humanos , Intestinos/metabolismo , Masculino , Organoides/metabolismo , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
5-Fluorouracil (5-FU) is a widely used chemotherapeutical that induces acute toxicity in the small and large intestine of patients. Symptoms can be severe and lead to the interruption of cancer treatments. However, there is limited understanding of the molecular mechanisms underlying 5-FU-induced intestinal toxicity. In this study, well-established 3D organoid models of human colon and small intestine (SI) were used to characterize 5-FU transcriptomic and metabolomic responses. Clinically relevant 5-FU concentrations for in vitro testing in organoids were established using physiologically based pharmacokinetic simulation of dosing regimens recommended for cancer patients, resulting in exposures to 10, 100 and 1000 µM. After treatment, different measurements were performed: cell viability and apoptosis; image analysis of cell morphological changes; RNA sequencing; and metabolome analysis of supernatant from organoids cultures. Based on analysis of the differentially expressed genes, the most prominent molecular pathways affected by 5-FU included cell cycle, p53 signalling, mitochondrial ATP synthesis and apoptosis. Short time-series expression miner demonstrated tissue-specific mechanisms affected by 5-FU, namely biosynthesis and transport of small molecules, and mRNA translation for colon; cell signalling mediated by Rho GTPases and fork-head box transcription factors for SI. Metabolomic analysis showed that in addition to the effects on TCA cycle and oxidative stress in both organoids, tissue-specific metabolic alterations were also induced by 5-FU. Multi-omics integration identified transcription factor E2F1, a regulator of cell cycle and apoptosis, as the best key node across all samples. These results provide new insights into 5-FU toxicity mechanisms and underline the relevance of human organoid models in the safety assessment in drug development.
Asunto(s)
Colon/efectos de los fármacos , Fluorouracilo/toxicidad , Intestino Delgado/efectos de los fármacos , Modelos Biológicos , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colon/patología , Relación Dosis-Respuesta a Droga , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/farmacocinética , Humanos , Intestino Delgado/patología , Masculino , Metabolómica , Organoides/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , TranscriptomaRESUMEN
Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.
Asunto(s)
Neoplasias de la Mama , Reprogramación Celular , Sistemas de Liberación de Medicamentos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , HumanosRESUMEN
Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Organoides/efectos de los fármacos , Tanquirasas/antagonistas & inhibidores , Adulto , Animales , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/patología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Organoides/patologíaRESUMEN
Antibody-drug conjugates (ADCs) offer a combination of antibody therapy and specific delivery of potent small-molecule payloads to target cells. The properties of the ADC molecule are determined by the balance of its components. The efficacy of the payload component increases with higher drug-to-antibody ratio (DAR), while homogeneous DAR = 8 ADCs are easily prepared by conjugation to the four accessible antibody hinge cystines. However, use of hydrophobic payloads has permitted only DAR = 2-4, due to poor pharmacokinetics and aggregation problems. Here, we describe generation and characterization of homogeneous DAR = 8 ADCs carrying a novel auristatin ß-D-glucuronide, MMAU. The glycoside payload contributed to overall hydrophilicity of the ADC reducing aggregation. Compared to standard DAR = 2-4 ADCs, cytotoxicity of the homogeneous DAR = 8 ADCs was improved to low-picomolar IC50 values against cancer cells in vitro. Bystander efficacy was restored after ADC internalization and subsequent cleavage of the glycoside, although unconjugated MMAU was relatively non-toxic to cells. DAR = 8 MMAU ADCs were effective against target antigen-expressing xenograft tumors. The ADCs were also studied in 3D in vitro patient-derived xenograft (PDX) assays where they outperformed clinically used ADC. In conclusion, increased hydrophilicity of the payload contributed to the ADC's hydrophilicity, stability and safety to non-target cells, while significantly improving cytotoxicity and enabling bystander efficacy.
RESUMEN
Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis and generally activated during toxicity in a chemical-specific manner. Here, we established a platform containing a panel of distinct adaptive stress response reporter cell lines based on BAC-transgenomics GFP tagging in HepG2 cells. Our current panel of eleven BAC-GFP HepG2 reporters together contains (1) upstream sensors, (2) downstream transcription factors and (3) their respective target genes, representing the oxidative stress response pathway (Keap1/Nrf2/Srxn1), the unfolded protein response in the endoplasmic reticulum (Xbp1/Atf4/BiP/Chop) and the DNA damage response (53bp1/p53/p21). Using automated confocal imaging and quantitative single-cell image analysis, we established that all reporters allowed the time-resolved, sensitive and mode-of-action-specific activation of the individual BAC-GFP reporter cell lines as defined by a panel of pathway-specific training compounds. Implementing the temporal pathway activity information increased the discrimination of training compounds. For a set of >30 hepatotoxicants, the induction of Srxn1, BiP, Chop and p21 BAC-GFP reporters correlated strongly with the transcriptional responses observed in cryopreserved primary human hepatocytes. Together, our data indicate that a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses, thus assisting in the future mechanism-based safety assessment of chemicals.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Cromosomas Artificiales Bacterianos , Proteínas Fluorescentes Verdes/análisis , Imagen Molecular/métodos , Pruebas de Toxicidad/métodos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Hep G2/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/toxicidad , Transporte Activo de Núcleo Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Biología Computacional , Bases de Datos Genéticas , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/metabolismo , Hígado/patología , Factor 2 Relacionado con NF-E2/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factor de Transcripción ReIA/genética , TransfecciónRESUMEN
BACKGROUND: Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets. METHODS: ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin. RESULTS: Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model. CONCLUSIONS: Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.
Asunto(s)
Antineoplásicos/farmacología , Productos Biológicos/farmacología , Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Neoplasias , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Visualización de Superficie Celular , Cisplatino/farmacología , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Biblioteca de Péptidos , Fenotipo , Anticuerpos de Cadena Única/farmacología , Esferoides Celulares , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Drug-induced liver injury (DILI) is an important clinical problem. Here, we used a genomics approach to in detail investigate the hypothesis that critical drug-induced toxicity pathways act in synergy with the pro-inflammatory cytokine tumor necrosis factor α (TNFα) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of â¼80 DILI compounds in primary human hepatocytes. Compounds displaying weak or no TNFα synergism, namely ketoconazole, nefazodone, and methotrexate, failed to synchronously induce both pathways. The ER stress induced was primarily related to protein kinase R-like ER kinase (PERK) and activating transcription factor 4 (ATF4) activation and subsequent expression of C/EBP homologous protein (CHOP), which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision-cut human liver slices. Targeted RNA interference studies revealed that whereas ER stress signaling through inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNFα-induced apoptosis. Whereas inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNFα cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNFα-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing toward caspase-8-dependent TNFα-induced apoptosis.
Asunto(s)
Carbamazepina/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Diclofenaco/toxicidad , Estrés Oxidativo/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Sinergismo Farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/inmunología , Transcriptoma/efectos de los fármacos , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate losing their hepatocyte functions and metabolic competence. We have developed a three-dimensional in vitro model using extracellular matrix-based hydrogel for long-term culture of the human hepatoma cell line HepG2. HepG2 cells cultured in this model stop proliferating, self-organize and differentiate to form multiple polarized spheroids. These spheroids re-acquire lost hepatocyte functions such as storage of glycogen, transport of bile salts and the formation of structures resembling bile canaliculi. HepG2 spheroids also show increased expression of albumin, urea, xenobiotic transcription factors, phase I and II drug metabolism enzymes and transporters. Consistent with this, cytochrome P450-mediated metabolism is significantly higher in HepG2 spheroids compared to monolayer cultures. This highly differentiated phenotype can be maintained in 384-well microtiter plates for at least 28 days. Toxicity assessment studies with this model showed an increased sensitivity in identifying hepatotoxic compounds with repeated dosing regimens. This simple and robust high-throughput-compatible methodology may have potential for use in toxicity screening assays and mechanistic studies and may represent an alternative to animal models for studying DILI.
Asunto(s)
Células Hep G2/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Toxicidad/métodos , Albúminas/metabolismo , Canalículos Biliares/efectos de los fármacos , Canalículos Biliares/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Inactivación Metabólica/genética , Hígado/metabolismo , Esferoides Celulares , Urea/metabolismoRESUMEN
Nephrotoxicity remains one of the main reasons for post-market drug withdrawal. Tumour necrosis factor α (TNF-α) secretion has been shown to underlie the nephrotoxicity induced by some of these drugs. Yet, there is currently no reliable and sensitive in vitro assay available to screen for nephrotoxicants of which toxicity largely depends on TNF-α secretion. Therefore, we developed and applied a sensitive fluorescence-based in vitro assay for TNF-α-mediated nephrotoxicity screening using mouse immortalized proximal tubular epithelial cells (IM-PTECs). Our assay allows rapid evaluation of TNF-α-mediated toxicant-induced apoptosis and necrosis using fixed endpoint and live cell measurements. To evaluate our assay, sixteen nephrotoxicants and two control non-nephrotoxicants were used. Out of the sixteen nephrotoxicants, eight induced cell death, of which five induced apoptosis as well as necrosis. Moreover, TNF-α significantly enhanced apoptotic cell death induced by cisplatin, cyclosporine A, tacrolimus and azidothymidine. These nephrotoxicants are known to induce inflammation in vivo which has been linked to an enhancement of nephrotoxicity for cisplatin, cyclosporine A and tacrolimus, confirming the functionality of our assay. Overall, our assay allows rapid and sensitive measurement of apoptosis and necrosis induced by a combination of nephrotoxicants and inflammatory components such as TNF-α and can be used as an alternative assay for nephrotoxicity prediction in vitro.
Asunto(s)
Bioensayo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Células Epiteliales/efectos de los fármacos , Túbulos Renales Proximales/citología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Células Epiteliales/metabolismo , Ratones , Necrosis/inducido químicamenteRESUMEN
Cis-diamminedichloroplatinum(II) (cisplatin)-induced renal proximal tubular apoptosis is known to be preceded by actin cytoskeleton reorganization, in conjunction with disruption of cell-matrix and cell-cell adhesion. In the present study, we show that the proinflammatory cytokine tumor necrosis factor α (TNF-α) aggravated these cisplatin-induced F-actin and cell adhesion changes, which was associated with enhanced cisplatin-induced apoptosis of immortalized proximal tubular epithelial cells. TNF-α-induced RelB expression and lentiviral small hairpin RNA (shRNA)-mediated knockdown of RelB, but not other nuclear factor κB members, abrogated the synergistic apoptosis observed with cisplatin/TNF-α treatment to the level of cisplatin-induced apoptosis. This protective effect was associated with increased stress fiber formation, cell-matrix, and cell-cell adhesion in the shRNARelB (shRelB) cells during cisplatin/TNF-α treatment, mimicking an epithelial-to-mesenchymal phenotypic switch. Indeed, gene array analysis revealed that knockdown of RelB was associated with upregulation of several actin regulatory genes, including Snai2 and the Rho GTPase proteins Rhophilin and Rho guanine nucleotide exchange factor 3 (ARHGEF3). Pharmacological inhibition of Rho kinase signaling re-established the synergistic apoptosis induced by combined cisplatin/TNF-α treatment of shRelB cells. In conclusion, our study shows for the first time that RelB is required for the cisplatin/TNF-α-induced cytoskeletal reorganization and apoptosis in renal cells by controlling a Rho kinase-dependent signaling network.
Asunto(s)
Apoptosis/fisiología , Cisplatino/farmacología , Transición Epitelial-Mesenquimal/fisiología , Túbulos Renales Proximales/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Transcripción ReIB/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Actinas/genética , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Células Cultivadas , Sinergismo Farmacológico , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Ratones , FN-kappa B/genética , Transducción de Señal , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/genética , Fibras de Estrés/metabolismo , Factor de Transcripción ReIB/genética , Regulación hacia Arriba/efectos de los fármacos , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.
Asunto(s)
Acetaminofén/toxicidad , Hígado/efectos de los fármacos , Pruebas de Toxicidad/métodos , Acetaminofén/metabolismo , Acetaminofén/farmacocinética , Adenosina Trifosfato/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Femenino , Humanos , Hidroquinonas/metabolismo , Técnicas In Vitro , Isomerismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas/metabolismo , Ratas , Ratas Wistar , Especificidad de la EspecieRESUMEN
Cisplatin-induced nephrotoxicity is an important limiting factor for cisplatin use. Tumor necrosis factor-α (TNF-α) is known to contribute to cisplatin-induced nephrotoxicity by inducing an inflammatory process aggravating the primary injury, thereby resulting in acute kidney injury (AKI). The present study investigates the pathways synergistically activated by cisplatin and TNF-α responsible for TNF-α-enhanced cisplatin-induced renal cell injury. To do so, immortalized renal proximal tubular epithelial cells (IM-PTECs) were co-treated with TNF-α and cisplatin. Under these conditions, cisplatin induced dose-dependent apoptosis in IM-PTECs, which was significantly enhanced by TNF-α. Transcriptomic analysis revealed that cisplatin inhibited the typical TNF-α response and cisplatin/TNF-α treatment up-regulated cell death pathways while it down-regulated survival pathways compared to cisplatin alone. In concordance, the gene expression levels of kidney injury markers combined with activation of specific inflammatory mediators were enhanced by cisplatin/TNF-α treatment, resembling the in vivo cisplatin-induced nephrotoxicity response. Furthermore, combined cisplatin/TNF-α treatment inhibited NF-κB nuclear translocation and NF-κB-mediated gene transcription leading to enhanced and prolonged JNK and c-Jun phosphorylation. JNK sustained activation further inhibited NF-κB signaling via a feedback loop mechanism. This led to an alteration in the transcription of the NF-κB-induced anti-apoptotic genes c-IAP2, Bcl-XL, Bruce and Bcl2 and pro-apoptotic genes Bfk and Xaf1 and consequently to sensitization of the IM-PTECs toward cisplatin/TNF-α-induced toxicity. In conclusion, our findings support a model whereby renal cells exposed to both cisplatin and TNF-α switch into a more pro-apoptotic and inflammatory program by altering their NF-κB/JNK/c-Jun balance.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antineoplásicos/efectos adversos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Transformada , Cisplatino/efectos adversos , Resistencia a Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/inmunología , Túbulos Renales Proximales/metabolismo , Ratones , FN-kappa B/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Nuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear translocation can vary significantly within a cell population and may dramatically change e.g. upon drug exposure. Furthermore, there is significant heterogeneity in individual cell response upon stress signaling. In order to systematically determine factors that define NF-κB translocation dynamics, high-throughput screens that enable the analysis of dynamic NF-κB responses in individual cells in real time are essential. Thus far, only NF-κB downstream signaling responses of whole cell populations at the transcriptional level are in high-throughput mode. In this study, we developed a fully automated image analysis method to determine the time-course of NF-κB translocation in individual cells, suitable for high-throughput screenings in the context of compound screening and functional genomics. Two novel segmentation methods were used for defining the individual nuclear and cytoplasmic regions: watershed masked clustering (WMC) and best-fit ellipse of Voronoi cell (BEVC). The dynamic NFκB oscillatory response at the single cell and population level was coupled to automated extraction of 26 analogue translocation parameters including number of peaks, time to reach each peak, and amplitude of each peak. Our automated image analysis method was validated through a series of statistical tests demonstrating computational efficient and accurate NF-κB translocation dynamics quantification of our algorithm. Both pharmacological inhibition of NF-κB and short interfering RNAs targeting the inhibitor of NFκB, IκBα, demonstrated the ability of our method to identify compounds and genetic players that interfere with the nuclear transition of NF-κB.
Asunto(s)
Núcleo Celular/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Imagen Molecular/métodos , FN-kappa B/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Algoritmos , Automatización , Forma de la Célula/efectos de los fármacos , Análisis por Conglomerados , Células Hep G2 , Humanos , Cinética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
UNLABELLED: Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase ß (IKKß) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity. CONCLUSION: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Diclofenaco/farmacología , Hepatocitos/metabolismo , Hepatocitos/patología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Apoptosis/fisiología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Caspasa 8/metabolismo , Línea Celular Tumoral , Inhibidores de la Ciclooxigenasa/farmacología , Sinergismo Farmacológico , Hepatocitos/efectos de los fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , MAP Quinasa Quinasa 4/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
In Drosophila oocytes, dorso-anterior transport of gurken mRNA requires both the Dynein motor and the heterogeneous nuclear ribonucleoprotein (hnRNP) Squid. We show that gurken transcripts are transported directly on microtubules by Dynein in nonmembranous electron-dense transport particles that also contain Squid and the transport cofactors Egalitarian and Bicaudal-D. At its destination, gurken mRNA is statically anchored by Dynein within large electron-dense cytoplasmic structures known as sponge bodies. Egalitarian and Bicaudal-D contribute only to active transport, whereas Dynein and Squid are also required for gurken mRNA anchoring and the integrity of sponge bodies. Disrupting Dynein function disperses gurken mRNA homogeneously throughout the cytoplasm, whereas the loss of Squid function converts the sponge bodies into active transport particles. We propose that Dynein acts as a static structural component for the assembly of gurken mRNA transport and anchoring complexes, and that Squid is required for the dynamic conversion of transport particles to sponge bodies.