RESUMEN
BACKGROUND: This phase Ib study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of the oral AKT inhibitor ipatasertib and chemotherapy or hormonal therapy in patients with advanced or metastatic solid tumors to determine combined dose-limiting toxicities (DLTs), maximum tolerated dose, and recommended phase II doses and schedules. PATIENTS AND METHODS: The clinical study comprised four combination treatment arms: arm A (with docetaxel), arm B [with mFOLFOX6 (modified leucovorin, 5-fluorouracil, and oxaliplatin)], arm C (with paclitaxel), and arm D (with enzalutamide). Primary endpoints were safety and tolerability; secondary endpoints were pharmacokinetics, clinical activity per Response Evaluation Criteria in Solid Tumors v1.1, and prostate-specific antigen levels. RESULTS: In total, 122 patients were enrolled. Common adverse events were diarrhea, nausea, vomiting, decreased appetite, and fatigue. The safety profiles of the combination regimens were consistent with those of the background regimens, except for diarrhea, hyperglycemia, and rash, which were previously observed with ipatasertib treatment. The only combination DLT across all treatment arms was one event of grade 3 dehydration (ipatasertib 600 mg and paclitaxel). Recommended phase II doses for ipatasertib were 600 mg (and mFOLFOX6) and 400 mg (and paclitaxel), respectively. The maximum assessed dose of ipatasertib 600 mg combined with docetaxel or enzalutamide was well tolerated. Coadministration with enzalutamide (a cytochrome P450 3A inducer) resulted in approximately 50% lower ipatasertib exposure. CONCLUSIONS: Ipatasertib in combination with chemotherapy or hormonal therapy was well tolerated with a safety profile consistent with that of ATP-competitive AKT inhibitors. CLINICAL TRIAL NUMBER: NCT01362374.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Humanos , Masculino , Dosis Máxima Tolerada , Neoplasias/tratamiento farmacológico , Piperazinas , Pirimidinas/uso terapéuticoRESUMEN
The concentration of meteoric 10Be in estuarine sediment samples has been measured by Spanish Accelerator for Radionuclides Analysis (SARA) at CNA and subsequently used to assess the denudation rate in Guadiana river basin together with the sediment budget method, on both sides of the frontier between Spain and Portugal. The two methods yielded coincident results. The estimation by the 10Be method gave the denudation rate of (0.76⯱â¯0.10)â¯×â¯10-2â¯cm/y. After correcting for an approximate 80% attenuation of the sediment discharge into the ocean, caused by the river dams, the sediment budget method yielded the rate of (0.77⯱â¯0.17)â¯×â¯10-2â¯cm/y.
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Berilio/análisis , Monitoreo de Radiación , Radioisótopos/análisis , Ríos/química , Contaminantes Radiactivos del Agua/análisis , Sedimentos Geológicos/química , EspañaRESUMEN
Tumor recurrence in glioblastoma (GBM) is, in part, attributed to increased epithelial-to-mesenchymal transition (EMT) and enhanced tumor cell dissemination in adjacent brain parenchyma after ionizing radiation (IR). EMT is associated with aggressive behavior, increased stem-like characteristics and treatment resistance in malignancies; however, the underlying signaling mechanisms that regulate EMT are poorly understood. We identified grade-dependent p21-activated kinases 4 (PAK4) upregulation in gliomas and further determined its role in mesenchymal transition and radioresistance. IR treatment significantly elevated expression and nuclear localization of PAK4 in correlation with induction of reactive oxygen species (ROS) and mesenchymal transition in GBM cells. Stable PAK4 overexpression promoted mesenchymal transition by elevating EMT marker expression in these cells. Of note, transcription factor-DNA-binding arrays and chromatin immunoprecipitation experiments identified the formation of a novel nuclear PAK4/PPARγ complex which was recruited to the promoter of Nox1, a peroxisome proliferator-activated receptor gamma (PPARγ) target gene. In addition, IR further elevated PAK4/PPARγ complex co-recruitment to Nox1 promoter, and increased Nox1 expression and ROS levels associated with mesenchymal transition in these cells. Conversely, specific PAK4 downregulation decreased PPARγ-mediated Nox1 expression and suppressed EMT in IR-treated cells. In vivo orthotopic tumor experiments showed inhibition of growth and suppression of IR-induced PPARγ and Nox1 expression by PAK4 downregulation in tumors. Our results provide the first evidence of a novel role for PAK4 in IR-induced EMT and suggest potential therapeutic efficacy of targeting PAK4 to overcome radioresistance in gliomas.
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Neoplasias Encefálicas/patología , Transición Epitelial-Mesenquimal/efectos de la radiación , Glioma/patología , NADPH Oxidasas/metabolismo , PPAR gamma/metabolismo , Quinasas p21 Activadas/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Femenino , Glioma/genética , Glioma/metabolismo , Humanos , Ratones , Ratones Desnudos , NADPH Oxidasa 1 , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/genética , PPAR gamma/genética , Regiones Promotoras Genéticas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Transfección , Quinasas p21 Activadas/genéticaRESUMEN
Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein that functions as an enzyme, cytokine, growth factor and hormone. As a target for oncology, NAMPT is particularly attractive, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. Inhibition of NAMPT generally results in NAD depletion, followed by ATP reduction and loss of cell viability. Herein, we describe NAMPT inhibitor (NAMPTi)-induced cardiac toxicity in rodents following short-term administration (2-7 days) of NAMPTi's. The cardiac toxicity was interpreted as a functional effect leading to congestive heart failure, characterized by sudden death, thoracic and abdominal effusion, and myocardial degeneration. Based on exposures in the initial in vivo safety rodent studies and cardiotoxicity observed, we conducted studies in rat and human in vitro cardiomyocyte cell systems. Based on those results, combined with human cell line potency data, we demonstrated the toxicity is both on-target and likely human relevant. This toxicity was mitigated in vitro by co-administration of nicotinic acid (NA), which can enable NAD production through the NAMPT-independent pathway; however, this resulted in only partial mitigation in in vivo studies. This work also highlights the usefulness and predictivity of in vitro cardiomyocyte assays using human cells to rank-order compounds against potency in cell-based pharmacology assays. Lastly, this work strengthens the correlation between cardiomyocyte cell viability and functionality, suggesting that these assays together may enable early assessment of cardiotoxicity in vitro prior to conduct of in vivo studies and potentially reduce subsequent attrition due to cardiotoxicity.
Asunto(s)
Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Animales , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/enzimología , Femenino , Compuestos Heterocíclicos con 2 Anillos/toxicidad , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/enzimología , Masculino , Nicotinamida Fosforribosiltransferasa/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Sulfonas/toxicidadRESUMEN
BACKGROUND AND AIMS: Inrathecal opioids like morphine added to local anaesthetic agents have been found to be effective in achieving prolonged post-operative analgesia. Intrathecal dexmedetomidine may be devoid of undesirable side effects related to morphine and hence, this study was designed to evaluate analgesic efficacy, haemodynamic stability and adverse effects of both these adjuvants in patients undergoing gynaecological surgeries. METHODS: This was a prospective, randomised, double blind study involving 25 patients in each group. Group M received 15 mg of 0.5% hyperbaric bupivacaine with 250 µg of morphine while Group D received 15 mg of 0.5% hyperbaric bupivacaine with 2.5 µg of dexmedetomidine. Characteristics of spinal block, time for first rescue analgesic and total dose of rescue analgesics were noted. Vital parameters and adverse effects were noted perioperatively. Data analysis was done with independent two sample t-test and Mann-Whitney U test. RESULTS: Time for first rescue analgesic (P = 0.056) and total analgesic demand were similar in both groups. Duration of sensory (P = 0.001) and motor (P = 000) block was significantly higher in dexmedetomidine group. Itching was noticed in 36% and nausea in 52% of patients in the morphine group, either of which was not seen in dexmedetomidine group. CONCLUSION: Intrathecal dexmedetomidine produces prolonged motor and sensory blockade without undesirable side effects but intraoperative hypotension was more frequent in dexmedetomidine group.
RESUMEN
Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation because of NRAS, KRAS, PTPN11, CBL and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3'-OH kinase (PI3K)/Akt cascades. Mx1-Cre, Kras(D12) and Mx1-Cre, Nf1(flox/)(-) mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, Kras(D12) mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients.
Asunto(s)
Compuestos Heterocíclicos con 3 Anillos/farmacología , Síndromes Mielodisplásicos/metabolismo , Trastornos Mieloproliferativos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas ras/genética , Animales , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Indazoles , Leucemia Mielomonocítica Crónica , Leucemia Mielomonocítica Juvenil , Sistema de Señalización de MAP Quinasas , Ratones , Síndromes Mielodisplásicos/tratamiento farmacológico , Trastornos Mieloproliferativos/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , SulfonamidasRESUMEN
The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
Asunto(s)
Compuestos de Anilina/farmacología , Apoptosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Neoplasias/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Ácidos Carboxílicos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Indoles/farmacología , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas/metabolismoAsunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Mieloma Múltiple/genética , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Sulfonamidas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.
Asunto(s)
Antígenos CD/genética , Epigénesis Genética , Glioma/genética , Glicoproteínas/genética , Células Madre Neoplásicas/metabolismo , Proteína Oncogénica p55(v-myc)/metabolismo , Péptidos/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Antígeno AC133 , Antígenos CD/metabolismo , Secuencia de Bases , Diferenciación Celular , Línea Celular Tumoral , Islas de CpG/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Glicoproteínas/metabolismo , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteína Oncogénica p55(v-myc)/inmunología , Péptidos/metabolismo , Plicamicina/farmacología , Análisis de Secuencia de ADN , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/inmunología , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.
Asunto(s)
Adenoviridae/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioma/genética , Glioma/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Adenoviridae/metabolismo , Animales , Apoptosis , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatología , Línea Celular Tumoral , Proliferación Celular , Femenino , Secciones por Congelación , Terapia Genética/instrumentación , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glioma/metabolismo , Glioma/fisiopatología , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéuticoRESUMEN
Ligation of CD40 on chronic lymphocytic leukemia (CLL) cells induces phenotypic and biochemical changes that facilitate CLL cell-T cell interactions and enhances the sensitivity of CLL cells to clearance by adaptive and innate immune-effector mechanisms. CLL cells can be transduced to express CD40 ligand (CD154) using a replication-defective adenovirus vector, thereby cross-linking CD40 on transduced and non-transduced, bystander CLL cells. In a previous study, patients received infusions of autologous CLL cells, transduced to express murine CD154 (mCD154), which induced anti-leukemic immune responses, but also anti-mCD154 antibodies. In this study, we report a phase I study, in which patients were infused with 1 × 10(8), 3 × 10(8) or 1 × 10(9) autologous CLL cells transduced ex vivo to express ISF35, a humanized, membrane-stable CD154. Infusions were well tolerated and consistently followed by reductions in blood lymphocyte counts and lymphadenopathy. After infusion, circulating CLL cells had enhanced or de novo expression of CD95, DR5, p73 and Bid, which enhanced their susceptibility to death-receptor-mediated or drug-induced apoptosis, including CLL cells with deletions at 17p13.1 (del(17p)). Two patients who had CLL with del(17p) had subsequent chemoimmunotherapy and responded well to treatment. In summary, infusions of autologous, ISF35-transduced CLL cells were well tolerated, had biological and clinical activity, and might enhance the susceptibility of CLL cells with del(17p) to chemoimmunotherapy.
Asunto(s)
Ligando de CD40/uso terapéutico , Terapia Genética/métodos , Leucemia Linfocítica Crónica de Células B/terapia , Adulto , Anciano , Antígenos CD40/inmunología , Ligando de CD40/efectos adversos , Cromosomas Humanos Par 17/genética , Femenino , Terapia Genética/efectos adversos , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Microscopía Fluorescente/métodos , Persona de Mediana Edad , Estadificación de Neoplasias , Eliminación de Secuencia , Receptor fas/inmunologíaRESUMEN
Nucleoside analogs are structurally similar antimetabolites that have a broad range of action and are clinically active in both solid tumors and hematological malignancies. Many of these agents are incorporated into DNA by polymerases during normal DNA synthesis, an action that blocks further extension of the nascent strand and causes stalling of replication forks. The molecular mechanisms that sense stalled replication forks activate cell cycle checkpoints and DNA repair processes, which may contribute to drug resistance. When replication forks are not stabilized by these molecules or when subsequent DNA repair processes are overwhelmed, apoptosis is initiated either by these same DNA damage sensors or by alternative mechanisms. Recently, strategies aimed at targeting DNA damage checkpoints or DNA repair processes have demonstrated effectiveness in sensitizing cells to nucleoside analogs, thus offering a means to elude drug resistance. In addition to their DNA synthesis-directed actions many nucleoside analogs trigger apoptosis by unique mechanisms, such as causing epigenetic modifications or by direct activation of the apoptosome. A review of the cellular and molecular responses to clinically relevant agents provides an understanding of the mechanisms that cause apoptosis and may provide rationale for the development of novel therapeutic strategies.
Asunto(s)
Apoptosis/efectos de los fármacos , Nucleósidos/farmacología , Transducción de Señal , Animales , Daño del ADN , Metilación de ADN , Reparación del ADN , Replicación del ADN , Humanos , Nucleósidos/química , Nucleósidos/metabolismoRESUMEN
Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.
Asunto(s)
Apoptosis , Glioma/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Glicoproteínas de Membrana/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Activación Enzimática , Humanos , MAP Quinasa Quinasa 4 , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
The mammalian cell cycle is exquisitely controlled by the cyclin-dependent kinases, which regulate cell cycle progression. Cell cycle transitions are, in turn, controlled by checkpoints that monitor the integrity and replication status of the genetic material before cells commit to either replicate or segregate their DNA. On activation, checkpoints interface with cyclin-Cdk complexes to block the cell cycle. Pharmacologic compounds that exploit our current knowledge of cell cycle and checkpoint pathway regulation offer insights into the development of novel therapeutic strategies.
Asunto(s)
Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN , Diseño de Fármacos , Animales , Daño del ADN , Humanos , Mamíferos , Neoplasias/tratamiento farmacológicoRESUMEN
Cyr61, a member of the CCN (CTGF/Cyr61/NOV) family of growth regulators, is a secreted cysteine-rich proangiogenic factor that has been implicated in tumorigenesis. Previous studies have also demonstrated that Cyr61 is regulated by 17beta-estradiol (E(2)) in the uterus. Therefore, we hypothesized that hormonal regulation of Cyr61 may be important in estrogen-dependent pathogenic processes such as breast tumorigenesis. Our study demonstrates that both Cyr61 messenger RNA and protein are induced by E(2) in MCF-7 mammary adenocarcinoma cells that primarily overexpress estrogen receptor alpha (ERalpha) in a dose-dependent and immediate early fashion. Cyr61 gene induction by E(2) is transcriptionally regulated by ERalpha as the antiestrogen, ICI 182,780, and actinomycin D blocked induction completely. In addition, Cyr61 is up-regulated in MCF-7 cells by epidermal growth factor (EGF) in an immediate early fashion as well. The functional relevance of steroid induction of Cyr61 in breast cancer cell growth is demonstrated by anti-Cyr61 neutralizing antibodies, which diminished E(2) and EGF-dependent DNA synthesis and dramatically reduced E(2)-driven cell proliferation by more than 70%. Most importantly, Cyr61 is overexpressed in 70% (28 of 40) of breast cancer patients with infiltrating ductal carcinoma and is localized exclusively to hyperplastic ductal epithelial cells. Moreover, the levels of Cyr61 protein are higher in breast tumors that are ER(+)/EGF receptor(+) than those that are ER(-)/EGF receptor(+), suggesting that estrogens may mediate Cyr61 expression in vivo. Collectively, our data suggest that Cyr61 may play a critical role in estrogen- as well as growth factor-dependent breast tumor growth.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , División Celular , Estradiol/análogos & derivados , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/genética , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular , Adenocarcinoma/genética , Adenocarcinoma/patología , Anticuerpos/farmacología , Proteína 61 Rica en Cisteína , ADN de Neoplasias/biosíntesis , Dactinomicina/farmacología , Factor de Crecimiento Epidérmico/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno , Fulvestrant , Sustancias de Crecimiento/inmunología , Sustancias de Crecimiento/fisiología , Humanos , Proteínas Inmediatas-Precoces/inmunología , Proteínas Inmediatas-Precoces/fisiología , Hibridación in Situ , ARN Mensajero/análisis , Receptores de Estrógenos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Uterine leiomyomas are the most common tumors of the reproductive tract, afflicting women between the ages of 30--55 yr. Although considered to be the leading cause of hysterectomies in the United States, little is known of the etiology and mechanisms of pathogenesis in leiomyomas. Accordingly, rapid analysis of differential expression (RADE) was employed to identify genes that are abnormally expressed in leiomyomas. Of the several genes identified, Cyr61, a member of the CCN family of growth and angiogenic regulators, was shown to be markedly down-regulated at the messenger ribonucleic acid (mRNA) and protein levels in leiomyoma tumors compared with the matched uterine myometrial controls (n = 38). In addition, in situ hybridization experiments corroborated the lack of Cyr61 expression in leiomyoma cells, whereas abundant transcript levels were identified in adjacent myometrial smooth muscle cells. To elucidate the mechanisms of Cyr61 gene regulation in leiomyomas, we determined the effects of ovarian steroids, basic fibroblast growth factor (bFGF), and serum, on Cyr61 expression using an ex vivo culture system. Treatment of human myometrial explants with 17 beta-estradiol and bFGF up-regulated Cyr61 transcripts, whereas the progesterone receptor agonist, R5020 (alone or in combination with 17 beta-estradiol), had no effect. Paradoxically, neither 17 beta-estradiol nor bFGF was capable of up-regulating Cyr61 mRNA in leiomyoma explants despite elevated levels of ER alpha mRNA, suggesting a possible defect in steroid and growth factor regulation. Thus, dysregulation of Cyr61 by estrogen and bFGF may contribute to down-regulation of Cyr61 in leiomyomas, which, in turn, may predispose uterine smooth muscle cells toward sustained growth.
Asunto(s)
Estradiol/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Sustancias de Crecimiento/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Leiomioma/metabolismo , Neoplasias Uterinas/metabolismo , Adulto , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Proteína 61 Rica en Cisteína , Regulación hacia Abajo , Estradiol/farmacología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Sustancias de Crecimiento/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Leiomioma/patología , Persona de Mediana Edad , Músculo Liso/metabolismo , Músculo Liso/patología , Miometrio/citología , Miometrio/metabolismo , Proteína Hiperexpresada del Nefroblastoma , ARN Mensajero/metabolismo , Valores de Referencia , Distribución Tisular , Regulación hacia Arriba , Neoplasias Uterinas/patologíaRESUMEN
The mechanisms of resistance to nucleoside analogues established in preclinical models are rarely found in primary tumors resistant to therapy with these agents. We tested the hypothesis that cells sense sublethal incorporation of analogues into DNA during replication and react by arresting further DNA synthesis and cell cycle progression. After removal of drug, cells may be able to repair damaged DNA and continue proliferation, thus escaping nucleoside analogue toxicity. As a corollary, we evaluated whether dysregulation of this mechanism causes cell death. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, we found that DNA synthesis decreased, cells arrested in S-phase transit, and 60-70% of the population accumulated in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium. S-phase-arrested cells were then treated with otherwise nontoxic concentrations of UCN-01, which caused rapid onset of apoptosis without cell cycle progression specifically in cells with an S-phase DNA content. Thus, S-phase arrest by nucleoside analogues sensitizes cells to UCN-01, which appears to activate signaling for death mechanisms and/or inhibit survival pathways. These results differ from those in cells arrested at the G2 checkpoint, in which UCN-01 abrogates cell cycle arrest, permitting cells to progress in the cell cycle before apoptosis.
Asunto(s)
Alcaloides/farmacología , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Desoxicitidina/farmacología , Fase S/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Estaurosporina/análogos & derivados , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Activation of the c-Jun NH2-terminal kinase type 1 (JNK1) signaling pathway is often associated with apoptosis. In this report, we elucidated the role of this kinase in the programmed cell death induced by the nucleoside analogue 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A). Treatment of ML-1 cells with 3 or 10 microM F-ara-A specifically killed cells in the S-phase of the population. Incorporation of F-ara-ATP, the nucleoside triphosphate of F-ara-A, into DNA resulted in the activation of JNK1 in a time- and dose-dependent fashion. Activation of JNK1 temporally preceded DNA fragmentation. When incorporation of F-ara-A into DNA was blocked by pretreatment of the cells with aphidicolin to inhibit DNA synthesis, neither JNK1 signaling nor apoptosis was evident. Furthermore, inhibition of JNK1 by treatment of the cells with forskolin or by pretreatment with an antisense oligonucleotide directed against JNK1 mRNA resulted in a decrease in F-ara-A-induced apoptosis. Finally, the JNK1 signaling pathway appeared to be upstream to that of the effector caspases in nucleoside analogue-induced apoptosis. Thus, our data strongly suggest that JNK1 is involved in transduction of F-ara-A-induced distress signals into an apoptotic response.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/fisiología , Daño del ADN/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfato de Vidarabina/análogos & derivados , Vidarabina/análogos & derivados , Vidarabina/farmacología , Apoptosis/efectos de los fármacos , Arabinonucleotidos/metabolismo , Caspasas/metabolismo , ADN/efectos de los fármacos , ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Leucemia Mieloide , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fase S/fisiología , Células Tumorales Cultivadas , Células U937 , Vidarabina/metabolismo , Fosfato de Vidarabina/metabolismoRESUMEN
Cytokine effects on immunity and inflammation often depend on the transcription factors termed signal transducers and activators of transcription (STATs), so STAT signaling pathways are candidates for influencing inflammatory disease. We reasoned that selective IFN responsiveness of the first STAT family member (Stat1) and Stat1-dependent immune-response genes such as intercellular adhesion molecule-1 (ICAM-1), IFN regulatory factor-1 (IRF-1), and Stat1 itself in airway epithelial cells provides a basis for detecting cytokine signaling abnormalities in inflammatory airway disease. On the basis of nuclear localization and phosphorylation, we found that epithelial Stat1 (but not other control transcription factors) was invariably activated in asthmatic compared with normal control or chronic bronchitis subjects. Furthermore, epithelial levels of activated Stat1 correlated with levels of expression for epithelial ICAM-1, IRF-1, and Stat1, and in turn, ICAM-1 levels correlated with T-cell accumulation in tissue. However, only low levels of IFN-gamma or IFN-gamma-producing cells were detected in airway tissue in all subjects. The results therefore provide initial evidence linking abnormal behavior of STAT pathways for cytokine signaling to the development of an inflammatory disease. In that context, the results also change the current scheme for asthma pathogenesis to one that must include a localized gain in transcriptional signal ordinarily used for a T helper 1-type cytokine (IFN-gamma) in combination with allergy-driven overproduction of T helper 2-type cytokines.