RESUMO
Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.
Assuntos
Antineoplásicos/uso terapêutico , Aurora Quinase A/antagonistas & inibidores , Mitose/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HeLa , Humanos , MasculinoRESUMO
Marked species-specific responses to agonists of the peroxisome proliferator-activated alpha receptor (PPAR alpha) have been observed in rats and dogs, two species typically used to assess the potential human risk of pharmaceuticals in development. In this study, we used primary cultured rat and dog hepatocytes to investigate the underlying mechanisms of a novel PPAR alpha and -gamma coagonist, LY465608, relative to fenofibrate, a prototypical PPAR alpha agonist. As expected, rat hepatocytes incubated with these two agonists demonstrated an increase in peroxisome number as evaluated by electron microscopy, whereas the peroxisome number remained unchanged in dog hepatocytes. Biochemical analysis showed that rat hepatocytes responded to PPAR agonists with an induction of both peroxisomal and mitochondrial beta-oxidation (PBox and MBox) activities. Dog hepatocytes treated with both PPAR agonists, however, did not show increased PBox activity but did demonstrate increased MBox activity. Analysis of peroxisomal beta-oxidation gene expression markers by quantitative real-time PCR confirmed that PPAR agonists induced the peroxisomal enzymes, acyl-coenzyme A (CoA) oxidase (Acox), enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (Ehhadh), and 3-ketoacyl-CoA thiolase (Acaa1) at the transcriptional level in rat hepatocytes, but not dog hepatocytes. Expression of mRNA for the mitochondrial beta-oxidation gene hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase (Hadhb), however, increased in both rat and dog hepatocytes, consistent with biochemical measurements of peroxisomal and mitochondrial beta-oxidation. Repeat-dose nonclinical safety studies of LY465608 revealed abnormities in mitochondrial morphology and evidence of single-cell necrosis following 30 days of dosing exclusively in dogs, but not in rats. Microarray analysis indicated that dog hepatocytes, but not rat hepatocytes, treated with LY465608 had an expression profile consistent with abnormalities in the regulation of cell renewal and death, oxidative stress, and mitochondrial bioenergetics, which may explain the canine-specific toxicity observed in vivo with this compound. This increased sensitivity to mitochondrial toxicity of canine hepatocytes relative to rat hepatocytes identified using gene expression was confirmed using the fluorescent indicator tetramethylrhodamine ethyl ester (TMRE) and flow cytometry. At doses of 0.1 microM LY465608, canine hepatocytes showed a greater shift in fluorescence indicative of mitochondrial damage than observed with rat hepatocytes treated at 10 microM. In summary, using rat and dog primary hepatocytes, we replicated the pharmacologic and toxicologic effects of LY465608 observed in vivo during preclinical development and propose an underlying mechanism for these species-specific effects.
Assuntos
Hepatócitos/efeitos dos fármacos , Compostos Orgânicos/farmacologia , PPAR alfa/agonistas , PPAR gama/agonistas , Animais , Bovinos , Células Cultivadas , Cães , Feminino , Fenofibrato/farmacologia , Fenofibrato/toxicidade , Citometria de Fluxo/métodos , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Hipolipemiantes/farmacologia , Hipolipemiantes/toxicidade , Masculino , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Compostos Orgânicos/toxicidade , Oxirredução , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Peroxissomos/ultraestrutura , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieRESUMO
Genotoxic stress causes a variety of cellular and molecular responses in mammalian cells, including cell cycle arrest, DNA repair, and apoptosis. These responses result from the interplay between the genotoxic events themselves, and the biological context in which they occur. To better understand this interplay, we investigated cytotoxicty, mutagenesis, cell cycle profile, and global gene expression in the human TK6 lymphoblastoid cell line exposed to six genotoxicants. The six compounds have broad structural diversity and cause genotoxic stress by many different mechanisms, including covalent modification (methyl methanesulfonate, mitomycin C), reactive oxygen species (hydrogen peroxide, bleomycin), and topoisomerase II inhibition (etoposide and doxorubicin). Cell cycle analysis was performed 4 and 20 h following a 4 h chemical exposure. Cells exposed to all compounds experienced S-phase arrest at the 8h time point, but by 24 h had markedly different cell cycle responses. Cells exposed to compounds that cause covalent modification had a strong G2/M arrest at 24 h. These cells also had a robust (>25-fold) increase in mutant frequency, and had a moderate but sustained p53 response at 4, 8, and 24h, detectable as approximately 2-5-fold increases in transcript levels for p21WAF1/CIP1, GADD45alpha, BTG2, and cyclin G1. In contrast, cells exposed to the reactive oxygen compounds had little or no G2/M arrest at 24 h and no increase in mutant frequency. In addition, these compounds caused a strong but transient induction of the p53 pathway, detectable as 15-25-fold increases in p21WAF1/CIP1 transcription at 4 h that decreased dramatically by 8h and was near control levels at 24 h. Thus, the mutagenic effect of compounds was consistent with G2/M arrest and sustained kinetics of p53 pathway activation. Global gene expression data were also consistent with the mutagenesis data. Activation of genes associated with cell cycle arrest, the p53 and TNF-related pathways, and chemokines and chemokine receptors, were particularly evident for the reactive oxygen compounds. In contrast, the most mutagenic compounds caused fewer and less robust changes in global gene expression. There was therefore an inverse relationship between global gene expression and mutagenic potency.
Assuntos
Ciclo Celular/efeitos dos fármacos , Dano ao DNA , Genoma Humano , Linfócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Linhagem Celular Tumoral , Fase G2 , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes p53/efeitos dos fármacos , Humanos , Cinética , Linfócitos/citologia , Linfócitos/fisiologia , Análise em Microsséries , Modelos Biológicos , Fase S , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
The purpose of this study was to examine the predictive value of humoral and cellular immune parameters in determining the immunotoxic effects of the oral administration of azathioprine (AZA), cyclophosphamide (CY), or cyclosporin A (CsA) at doses of 25/17, 10, or 25 mg/kg per day, respectively, for 30 days in F344 female rats. The effect of these known immunosuppressive compounds on the immune response was assessed in a humoral model that consisted of the administration of nitrophenyl-chicken gamma globulin (NP-CgammaG) and keyhole limpet hemocyanin (KLH) antigens during immunosuppressive treatment and the measurement of resulting rat antigen-specific IgG and IgM, as well as total IgG, levels. Cellular assessment parameters were collected from the same groups of animals as the humoral parameters and included organ weights and cellularity, hematology, lymphocyte phenotype characteristics, spleen cell mitogen stimulation (T and B cell-dependent), splenic natural killer (NK) cell cytotoxicity, and bone marrow cellularity and lymphocyte phenotype differential. Although decreases in several of the cellular assay parameters were observed, the only functional assays to demonstrate a statistically significant immunosuppressive effect by all three immunosuppressive agents were the antigen-specific serum IgG levels. The primary (day 10; 15 days post-immunization) and secondary (day 25; 5 days post-rechallenge) nitrophenyl (NP) responses were significantly suppressed by > or =60%. The use of NP hapten provided consistent responses when analyzed with a sensitive, well developed, ELISA methodology. Absolute lymphocyte phenotyping and lymphocyte hematology were also predictive of T cell immunosuppression for all three compounds. The data presented herein suggests that these two parameters, NP-IgG humoral response and lymphocyte phenotyping, are sufficient for identifying immunosuppressive compounds.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Hemocianinas/imunologia , Imunidade Celular/efeitos dos fármacos , Imunossupressores/toxicidade , Nitrofenóis/imunologia , Animais , Formação de Anticorpos/imunologia , Azatioprina/toxicidade , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Ciclofosfamida/toxicidade , Ciclosporina/toxicidade , Feminino , Haptenos/imunologia , Imunidade Celular/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Modelos Animais , Ratos , Ratos Endogâmicos F344 , Baço/efeitos dos fármacos , Baço/imunologia , Timo/efeitos dos fármacos , Timo/imunologia , Testes de Toxicidade/métodosAssuntos
Carcinógenos/toxicidade , Hidrazinas/toxicidade , Fígado/efeitos dos fármacos , Xenobióticos/toxicidade , Alanina Transaminase/antagonistas & inibidores , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/antagonistas & inibidores , Aspartato Aminotransferases/metabolismo , Carcinógenos/administração & dosagem , Relação Dose-Resposta a Droga , Hidrazinas/administração & dosagem , Fígado/enzimologia , Masculino , Fosfato de Piridoxal/metabolismo , Ratos , Ratos Sprague-Dawley , Xenobióticos/administração & dosagemRESUMO
Phospholipidosis, or intracellular accumulation of phospholipids, is caused by specific classes of xenobiotics. This phenomenon represents a challenge for risk assessment that could benefit from the use of biomarkers in the clinical development of new drug candidates. Flow cytometry, coupled with the lipophilic fluoroprobe Nile red, was correlated to histopathology, electron microscopy and inorganic phosphorus detection to validate the utility of this method for monitoring phospholipidosis in peripheral blood leukocytes. Replicate studies with model test compounds were conducted in which F344 rats were given 4 or 7 doses of either maprotiline hydrochloride, imipramine hydrochloride, tilorone dihydrochloride, amikacin hydrate or vehicle control. Transmission electron and light microscopy were used to examine peripheral blood smears and tissue samples for the presence of cytoplasmic vacuoles. Unstained and Nile red stained lysed peripheral blood samples were acquired for analysis using a FACScan flow cytometer. Inorganic phosphorus concentration in the liver was determined from extracted phospholipids and compared with flow cytometry and histological data. It was demonstrated that flow cytometric analysis of Nile red stained lysed whole blood can be used to detect drug-induced phospholipid accumulation in circulating peripheral leukocytes. Furthermore, clinically detectable leukocyte phospholipidosis may be a useful surrogate for coincident or premonitory detection of target organ phospholipidosis.