RESUMEN
Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl(+) leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl(+) stem cells is needed to cure leukemias caused by Bcr-Abl(+) cells. Human Bcr-Abl(+) cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl(+) sublines by selection in long-term hypoxic cultures (1.0% O(2)). Interestingly, HA-Bcr-Abl(+) cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher beta-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl(+) cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl(+) cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl(+) leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl(+) leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment.
Asunto(s)
Lactoilglutatión Liasa/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Animales , Benzamidas , Hipoxia de la Célula , Línea Celular Tumoral , Dasatinib , Humanos , Mesilato de Imatinib , Lactoilglutatión Liasa/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Ratones , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacología , Trasplante Heterólogo , beta Catenina/metabolismoRESUMEN
Although wild-type p53 protein is overexpressed in first trimester trophoblast, it is inactive towards its target genes Metalloproteinase 2 and 9. This seems to be due to a complex mechanism of inactivation and stabilization of p53 relying on the formation of protein complexes involving the N-terminus of p53. To detect the proteins associated with this sequence, we incubated biotinylated p53 N-terminal peptide in cytotrophoblastic cell medium 24 h before lysis of cells. We purified the proteins retained on biotinylated peptide using a neutravidin affinity column. Proteins were then identified by peptide mass finger printing followed or not by peptide fragmentation sequencing. Among these proteins, we identified glucose-regulated protein 78 (GRP78) and verified its interaction with p53 in trophoblastic cells by immunoprecipitation and Western blot analysis. Moreover, the decreased expression of GRP78 induced by GRP78siRNA or versipelostatin decreased the formation of high molecular weight p53 complexes and p53 monomer and increased trophoblastic invasion. These results suggest that GRP78 is involved in inactivation and stabilization of p53 and in the regulation of trophoblastic invasion.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Trofoblastos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/análisis , Humanos , Datos de Secuencia Molecular , Placenta/citología , Embarazo , Unión Proteica , Mapeo de Interacción de Proteínas , Trofoblastos/citología , Proteína p53 Supresora de Tumor/análisisRESUMEN
Enhanced lipid biosynthesis is a characteristic feature of cancer. Deregulated lipogenesis plays an important role in tumour cell survival. These observations suggest that enzymes in the lipid synthesis pathway would be rational therapeutic targets for cancer. To this end, we review the enzymes in de novo fatty-acid synthesis and related pathways.
Asunto(s)
Antineoplásicos/uso terapéutico , Diseño de Fármacos , Ácidos Grasos/biosíntesis , Neoplasias/tratamiento farmacológico , ATP Citrato (pro-S)-Liasa/metabolismo , Coenzima A Ligasas/metabolismo , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/enzimologíaRESUMEN
Extracellular acidosis (low pH) is a tumor microenvironmental stressor that has a critical function in the malignant progression and metastatic dissemination of tumors. To survive under stress conditions, tumor cells must evolve resistance to stress-induced toxicity. Acyl-CoA synthetase 5 (ACSL5) is a member of the ACS family, which converts fatty acid to acyl-CoA. ACSL5 is frequently overexpressed in malignant glioma, whereas its functional significance is still unknown. Using retrovirus-mediated stable gene transfer (gain of function) and small interfering RNA-mediated gene silencing (loss of function), we show here that ACSL5 selectively promotes human glioma cell survival under extracellular acidosis. ACSL5 enhanced cell survival through its ACS catalytic activity. To clarify the genome-wide changes in cell signaling pathways by ACSL5, we performed cDNA microarray analysis and identified an ACSL5-dependent gene expression signature. The analysis revealed that ACSL5 was critical to the expression of tumor-related factors including midkine (MDK), a heparin-binding growth factor frequently overexpressed in cancer. Knockdown of MDK expression significantly attenuated ACSL5-mediated survival under acidic state. These results indicate that ACSL5 is a critical factor for survival of glioma cells under acidic tumor microenvironment, thus providing novel molecular basis for cancer therapy.
Asunto(s)
Acidosis/enzimología , Coenzima A Ligasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/enzimología , Glioma/patología , Catálisis , Línea Celular Tumoral , Supervivencia Celular/genética , Perfilación de la Expresión Génica , Glioma/genética , Humanos , Midkina , Proteínas de Neoplasias/genética , Factores de Crecimiento Nervioso/genética , Regulación hacia ArribaRESUMEN
Chemoresistance and therapeutic selectivity are major obstacles to successful chemotherapy of ovarian cancer. Manganese superoxide disumutase (MnSOD) is an important antioxidant enzyme responsible for the elimination of superoxide radicals. We reported here that MnSOD was significantly elevated in ovarian cancer cells and its overexpression was one of the mechanisms that increased resistance to apoptosis in cancer cells. Knockdown of MnSOD by small-interfering RNA (siRNA) led to an increase in superoxide generation and sensitisation of ovarian cancer cells to the two front-line anti-cancer agents doxorubicin and paclitaxel whose action involved free-radical generation. This synergistic effect was not observed in non-transformed ovarian surface epithelial cells. Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway. Evaluation of signalling pathways showed that MnSOD siRNA enhanced doxorubicin- and paclitaxel-induced phosphorylation of extracellular signal-regulated kinase 1/2. Akt activation was not affected. These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.
Asunto(s)
Caspasa 9/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Superóxido Dismutasa/antagonistas & inhibidores , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 9/genética , Inhibidores de Caspasas , Línea Celular Tumoral , Doxorrubicina/farmacología , Femenino , Humanos , Oligopéptidos/farmacología , Neoplasias Ováricas/genética , Paclitaxel/farmacología , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , TransfecciónRESUMEN
FOXO (Forkhead box O) transcription factors are involved in cell-cycle arrest or apoptosis induction by transcripting cell-cycle inhibitor p27(KIP1) or apoptosis-related genes, respectively. Akt/protein kinase B promotes cell proliferation and suppresses apoptosis, in part, by phosphorylating FOXOs. Phosphorylated FOXOs could not exhibit transcriptional activity because of their nuclear export. Here we show that p15(INK4b) and p19(INK4d) transcription is associated with FOXO-mediated G1 cell-cycle arrest. Inhibition of Akt signaling by PI3K inhibitors, a PDK1 inhibitor, or dominant-negative Akt transfection increased expression of p15(INK4b) and p19(INK4d) but not p16(INK4a) and p18(INK4c). Ectopic expression of wild type or active FOXO but not inactive form also increased p15(INK4b) and p19(INK4d) levels. FOXOs bound to promoter regions and induced transcription of these genes. No increase in the G1-arrested cell population, mediated by PI3K inhibitor LY294002, was observed in INK4b-/- or INK4d-/- murine embryonic fibroblasts. In summary, FOXOs are involved in G1 arrest caused by Akt inactivation via p15(INK4b) and p19(INK4d) transcription.
Asunto(s)
Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/genética , Factores de Transcripción Forkhead/fisiología , Regulación de la Expresión Génica/fisiología , Animales , Línea Celular , Cromonas/farmacología , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Fase G1/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Noqueados , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Inhibition of telomerase activity by telomerase inhibitors induces a gradual loss of telomeres, and this in turn causes cancer cells to enter to a crisis stage. Here, we report the telomerase inhibitor telomestatin, which is known to stabilize G-quadruplex structures at 3' single-stranded telomeric overhangs (G-tails), rapidly dissociates TRF2 from telomeres in cancer cells within a week, when given at a concentration that does not cause normal cells to die. The G-tails were dramatically reduced upon short-term treatment with the drug in cancer cell lines, but not in normal fibroblasts and epithelial cells. In addition, telomestatin also induced anaphase bridge formation in cancer cell lines. These effects of telomestatin were similar to those of dominant negative TRF2, which also causes a prompt loss of the telomeric G-tails and induces an anaphase bridge. These results indicate that telomestatin exerts its anticancer effect not only through inhibiting telomere elongation, but also by rapidly disrupting the capping function at the very ends of telomeres. Unlike conventional telomerase inhibitors that require long-term treatments, the G-quadruplex stabilizer telomestatin induced prompt cell death, and it was selectively effective in cancer cells. This study also identifies the TRF2 protein as a therapeutic target for treating many types of cancer which have the TRF2 protein at caps of the telomere DNA of each chromosome.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas Nucleares/metabolismo , Oxazoles/farmacología , Proteínas Similares a la Proteína de Unión a TATA-Box/metabolismo , Telómero , Anafase , Muerte Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales , Femenino , Fibroblastos , Células HeLa , Humanos , Telómero/ultraestructura , Proteína 2 de Unión a Repeticiones Teloméricas , Células Tumorales CultivadasRESUMEN
Peritoneal dissemination is the most frequent type of recurrence in patients with gastric cancer with serosal exposure, irrespective of whether they have undergone curative gastrectomy. The purpose of this study was to establish a method to detect micrometastatic cells in the abdominal cavity and predict peritoneal recurrence in patients with such gastric carcinomas. A total of 86 patients with gastric carcinoma, undergoing gastrectomy, were examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect carcinoembryonic antigen (CEA) mRNA in abdominal lavage fluid. Twenty-four cases without serosal exposure were negative, while all 13 cases with macroscopic peritoneal dissemination were positive for CEA mRNA. Among the 49 cases with macroscopic serosal invasion and without peritoneal metastasis, cancer cells were detected in 27 cases with RT-PCR while in only 6 cases with conventional cytology. All cytologically-positive cases were also positive for CEA mRNA. Among the 27 CEA-positive cases, 15 patients (56%) relapsed with peritoneal metastasis within 12 months after gastrectomy. In contrast, none of the 22 CEA-negative cases had peritoneal recurrence within 16-60 months of observation, whereas in 43 cytologically-negative cases, 10 patients relapsed with peritoneal recurrence. As compared with conventional cytological examination, this method would be clinically more beneficial for detecting free cancer cells in the peritoneal cavity and for predicting peritoneal recurrence in gastric carcinoma with serosal invasion.
Asunto(s)
Antígeno Carcinoembrionario/genética , Neoplasias Peritoneales/genética , ARN Mensajero/análisis , Neoplasias Gástricas/genética , Cavidad Abdominal , Secuencia de Bases , Cartilla de ADN , Femenino , Humanos , Masculino , Invasividad Neoplásica , Neoplasias Peritoneales/epidemiología , Neoplasias Peritoneales/mortalidad , Neoplasias Peritoneales/patología , Reacción en Cadena de la Polimerasa/métodos , Valor Predictivo de las Pruebas , ARN Mensajero/genética , Recurrencia , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Análisis de Supervivencia , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The management of advanced cancer presents the greatest challenge to physicians involved in oncology. There will usually be a large burden of disease; cure is unlikely; and the needs of the patient in terms of pain control and palliation will also be important over and above the direct treatment of the disease. Different issues will arise depending on the site and pathological type of the cancer. Increasingly over the past few years, treatment protocols and guidelines have been developed for different cancers, but these can only be rough guides rather than definite treatment recommendations. Additionally in most cancers advanced disease offers the opportunity for evaluation of new treatments in Phase II studies and other trials. With the new generation of molecular targeted therapies, such as EGFR inhibitors, striking results are being seen in advanced disease that compare favourably with what has been seen previously. Other agents such as those which attack the tumour vasculature may also have promise in this setting. Palliation is also an important aspect of the management of advanced disease, and pain control in particular is an important component of patient management. In summary, the treatment of advanced disease provides a test bed for new agents, but this need to develop better cancer therapies must be balanced against patient needs for a pain-free and comfortable end to life.
Asunto(s)
Neoplasias/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Femenino , Humanos , Masculino , Neoplasias/tratamiento farmacológico , Manejo del Dolor , Cuidados Paliativos , Calidad de VidaRESUMEN
It remains a question whether hematogeneous metastasis arises from a single cancer cell attached to the local endothelium or from a cluster of cancer cells trapped in the vascular bed in the target organ. Adhesive interaction of the single cell form and the clustered form of cancer cells was examined under flow conditions, using two subclones of mouse colon adenocarcinoma Colon 26. A subclone NL17, but not NL14, formed many clusters composed of tumor cells and platelets just after the addition of platelet rich plasma (PRP). Under the shear of 1.0 dyn/cm3, the clustered form of NL17 tethered on laminin or mouse endothelial cell line in hepatic sinusoids (HSE) more frequently than the single cell form of NL17 and NL14. However, all of the clusters showed only transient attachment and never underwent stable arrest on coated laminin, while the single cell form of NL14 and NL17 underwent immediate arrest under shear conditions. On HSE stimulated with TNF-alpha, a small number of NL17 clusters made stable adhesion, although all the clusters detached if the shear stress was increased above 4.0 dyn/cm2. In contrast, the single form of arrested NL17 as well as NL14 remained adherent even at shear of 8.0 dyn/cm2. Compared with single cell, binding of cancer cell clusters to laminin and HSE showed lower resistance to shear stress, although they had adhesive interactions more frequently in flow condition. Since NL17 cells form significantly more metastases by intravenous injection in vivo, our data suggest that "stable adhesion" observed in our flow assay system is not always a prerequisite for clustered cancer cells to develop into metastatic lesions.
Asunto(s)
Adhesión Celular/fisiología , Neoplasias del Colon/fisiopatología , Laminina/fisiología , Sarcoma Experimental/patología , Sarcoma Experimental/fisiopatología , Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Animales , Ciclo Celular/fisiología , Células Clonales , Neoplasias del Colon/patología , Ratones , Estrés Mecánico , Células Tumorales CultivadasRESUMEN
SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis, were examined for their reversing effects in vitro on various multidrug-resistant (MDR) tumor cells overexpressing P-glycoprotein. These two compounds in the range of 3-10 microM completely reversed the resistance of MDR variant cells, mouse leukemia P388 cells [vincristine (VCR)-resistant P388/VCR and adriamycin (ADM)-resistant P388/ADM], human myelogenous leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) and human ovarian cancer A2780 cells (ADM-resistant AD(10)), against VCR. Both compounds moderately potentiated the sensitivity of the MDR cells to ADM but the reversal was not complete. SNF4435C and D significantly increased the intracellular accumulation of VCR in AD(10) cells as potently as verapamil, cyclosporin A (CysA) and FK506, whereas the compounds exerted no effect on the accumulation of VCR in the drug-sensitive parent cells. Moreover, SNF4435C improved the chemotherapeutic efficacy of VCR in the treatment of P388/VCR-bearing mice. When 10 mg/kg SNF4435C was administered intraperitoneally to the mice concurrently with 0.2 mg/kg VCR for every 5 days, a treated/control (T/C) value of 143% was obtained. These results suggest that the compounds are useful candidates or tools for MDR modification in cancer chemotherapy.
Asunto(s)
Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Nitrocompuestos/farmacología , Pironas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Sinergismo Farmacológico , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Mitolactol/farmacología , Mitolactol/uso terapéutico , Mitomicinas/farmacología , Mitomicinas/uso terapéutico , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Células Tumorales Cultivadas , Vincristina/farmacología , Vincristina/uso terapéuticoRESUMEN
For cancer chemotherapy, avoiding the side effects of chemotherapeutic agents is difficult. Multidrug resistance is one of the major obstacles to successful cancer chemotherapy. P-Glycoprotein (P-gp) serves as an efflux pump and plays a key role in the multidrug resistance. We examined the effect of MRK-16, a monoclonal antibody against P-gp, modified liposomes (MRK-Lip) on the human myelogenous leukemia K-562 cells and its adriamycin resistance cell line K-562/ADM cells to avoid the side effects and to reverse the multidrug resistance. The uptake of vincristine (VCR) by K-562/ADM cells was lower than that by K-562 cells. This low uptake was increased in the presence of verapamil and MRK-16, however, it was not increased in the presence of control antibody, IgG2A. The binding of MRK-Lip to K-562/ADM cells was higher than that of IgG2A-modified liposome (IgG-Lip) and liposome without modification (Cont-Lip). Moreover, the cytotoxicity of VCR-encapsulated MRK-Lip to K-562/ADM cells was higher than that of VCR-encapsulated IgG-Lip and Cont-Lip. These results suggest that the interaction between liposomes and multidrug resistance cells was increased by the modification of liposomes with MRK-16. Consequently, the usefulness of MRK-Lip in cancer chemotherapy as a potent carrier was suggested.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Liposomas/administración & dosificación , Vincristina/administración & dosificación , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Células Tumorales Cultivadas , Vincristina/farmacocinética , Vincristina/farmacologíaRESUMEN
Under hypoxia, HIF-1alpha binds to aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-1beta) to activate expression of genes important for cell survival. Alternatively, HIF-1alpha can bind to the tumor suppressor p53 and promote p53-dependent apoptosis. Here we show that the opposite functions of HIF-1alpha are distinguished by its phosphorylation status. Two distinguishable forms of HIF-1alpha, phosphorylated and dephosphorylated, were induced during hypoxia-induced apoptosis. The phosphorylated HIF-1alpha was the major form that bound to ARNT. Ectopically expressed ARNT was consistently able to enhance HIF-1alpha phosphorylation in a binding-dependent manner. In contrast, the dephosphorylated HIF-1alpha was the major form that bound to p53. Depletion of the dephosphorylated HIF-1alpha, by using the Hsp90 inhibitor geldanamycin A that had little effect on the phosphorylated HIF-1alpha expression, suppressed p53 induction and subsequent apoptosis. Depletion of dephosphorylated HIF-1alpha also prevented hypoxia-induced nuclear accumulation of HDM2, a negative regulator of p53. Our results indicate that the functions of HIF-1alpha varied with its phosphorylation status and that dephosphorylated HIF-1alpha mediated apoptosis by binding to and stabilizing p53.
Asunto(s)
Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Hidrocarburo de Aril , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Translocador Nuclear del Receptor de Aril Hidrocarburo , Benzoquinonas , Caspasas/metabolismo , Núcleo Celular/metabolismo , Proteínas HSP90 de Choque Térmico/farmacología , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Lactamas Macrocíclicas , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Quinonas/farmacología , Proteína p53 Supresora de Tumor/efectos de los fármacosRESUMEN
In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.
Asunto(s)
Barrera Hematoencefálica , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Ácido gamma-Aminobutírico/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Capilares , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Línea Celular , Endotelio Vascular/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática , Semivida , Cinética , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Microinyecciones , Ácidos Nipecóticos/farmacología , Lóbulo Parietal/metabolismo , Probenecid/farmacología , Ratas , Ratas Sprague-Dawley , Taurina/farmacología , Tritio , beta-Alanina/farmacología , Ácido gamma-Aminobutírico/administración & dosificación , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
PURPOSE: Glyoxalase I (GLO1) is an enzyme that plays a role in the detoxification of methylglyoxal, a side-product of glycolysis. We previously reported that GLO1 was a resistant factor to antitumor agent-induced apoptosis, and that S-p-bromobenzylglutathione cyclopentyl diester (BBGC), an effective inhibitor of GLO1, selectively sensitized to etoposide the drug-resistant human leukemia cells that overexpressed GLO1. In this study, we quantitatively measured GLO1 enzyme activity in various human solid tumor cells, and the antiproliferative effect of the GLO1 inhibitor was examined. EXPERIMENTAL DESIGN: BBGC-induced apoptosis was assessed by flow cytometry. To evaluate antitumor activity of BBGC in vivo, we developed human cancer xenografts in nude mice. RESULTS: We found that GLO1 enzyme activity was higher in all of the 38 human cancer cell lines that we examined than in the normal tissue samples. Moreover, GLO1 activity was frequently elevated in human lung carcinoma cells. Positive correlation between cellular GLO1 activity and BBGC sensitivity was observed in the lung cancer cell lines. Human lung cancer NCI-H522 and DMS114 cells, expressing higher GLO1 activity, underwent apoptosis when treated with BBGC, whereas A549 cells, expressing lower activity, did not. BBGC induced the activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase 1 (JNK1) and p38 mitogen-activated protein kinase (MAPK), which led to caspase activation in GLO1-overexpressing tumor cells. BBGC significantly inhibited the growth of xenografted DMS114 and human prostate cancer DU-145. CONCLUSIONS: Our present results indicate that GLO1 is a tumor-specific target enzyme especially in human lung carcinoma cells and that the GLO1 inhibitor is a potent chemotherapeutic agent to repress GLO1-overexpressing human tumors.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutatión/farmacología , Lactoilglutatión Liasa/antagonistas & inhibidores , Neoplasias Pulmonares/prevención & control , Animales , Caspasas/efectos de los fármacos , Caspasas/metabolismo , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Glutatión/análogos & derivados , Humanos , Lactoilglutatión Liasa/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epithelial cells undergo death receptor-dependent apoptosis when detached from matrix, a process termed anoikis. Activation of Akt/protein kinase B (PKB) by matrix attachment protects cells from anoikis. In this study, we establish a link between anoikis and Akt/PKB-mediated survival by demonstrating that Akt/PKB is cleaved by caspases in matrix-detached epithelial cells by a mechanism that involves death receptors. Reduced levels of Akt/PKB protein were observed in detached Madin-Darby canine kidney cells relative to cells attached to collagen. Equivalent levels of Akt/PKB, however, were detected in matrix-adherent and detached cells after inhibition of caspase activity or expression of an Akt/PKB mutant (D108+119A) that is resistant to caspase cleavage. The contribution of death domain-containing proteins to Akt/PKB cleavage was evidenced by the ability of dominant negative Fas-associated death domain to restore normal levels of Akt/PKB in matrix-detached cells. Importantly, expression of a cleavage-resistant Akt/PKB mutant protected matrix-detached cells from apoptosis. These studies suggest that members of the death receptor family promote the caspase-mediated cleavage of Akt/PKB and that this event contributes to anoikis.
Asunto(s)
Apoptosis , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Anoicis , Caspasas/fisiología , Línea Celular , Perros , Proteínas Proto-Oncogénicas c-akt , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Miembro 25 de Receptores de Factores de Necrosis TumoralRESUMEN
Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Quinolinas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/secundario , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Etopósido/administración & dosificación , Etopósido/farmacología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones SCID , Metástasis de la Neoplasia , Quinolinas/administración & dosificación , Vincristina/administración & dosificación , Vincristina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA topoisomerase inhibitors are effective chemotherapeutic agents on several solid tumor cells. They induce a specific signaling cascade that executes an active cell death process (apoptosis), including caspase activation, and the blockage of the signaling is associated with drug-resistance of tumor cells. However, little is known about the initial signal transduction induced by the agents. In the present study, we screened genes that are initially upregulated in caspase-independent manner. We found that the activating transcription factor 3 (ATF3) protein, a repressor of cyclic-AMP responsive element (CRE)-dependent transcription, was strongly induced among CRE-BP/ATF members and subsequently accumulated in nuclei following camptothecin or etoposide treatment. During induction of apoptosis, the accumulation and the nuclear translocation of ATF3 coincided with the activation of caspase protease and were not inhibited by the broad caspase inhibitor Z-VAD-fmk, indicating that ATF3 induction is not a downstream event of caspase activation. When stably or transiently overexpressed, ATF3 markedly accelerated the drug-induced apoptosis and enhanced caspase protease activation. ATF3 strongly downregulated CRE-dependent transcription, while ATF3 did not affect the expression levels of Bcl-2, Bcl-x, or Bax. Our present results indicate that ATF3 plays a critical role in accelerating caspase protease activation and apoptosis. Since CRE-dependent transcription functions as cell survival signaling, ATF3 could control the upstream signaling of apoptosis by repressing CRE-dependent gene expression of cell survival factors.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 3 , Transporte Activo de Núcleo Celular/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Camptotecina/farmacología , Inhibidores de Caspasas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Fibrosarcoma/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inhibidores de Topoisomerasa I , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Cyclic hydroxamic-acid-containing peptide 1 (CHAP1), designed as a hybrid of trichostatin A and trapoxin, is a lead compound for the development of potent inhibitors of histone deacetylase (HDAC). In this study, we synthesized a series of CHAP derivatives and evaluated their biological activities by monitoring the potency of their inhibition of HDAC activity, their ability to augment the expression of MHC class-I molecules in B16/BL6 cells, and their effect on cell proliferation. A structure-activity relationship study using these three assay systems revealed several requirements of their structure for the strong inhibition of HDAC not only in the cell-free situation, but also in cells. When the structures of CHAP derivatives are represented as cyclo(-Asu(NHOH)-AA(2)-AA(3)-Pro or Pip-)(n), where Asu(NHOH) and Pip are zeta-hydroxamide-alpha-aminosuberic acid and pipecolic acid, respectively, (a) the tetrapeptide structure (n = 1) was better than the octapeptide one (n = 2); (b) AA(2) and AA(3) should be hydrophobic; and (c) the combination of amino acid chirality should be LDLD for the strongest inhibition of HDAC in cells (LDLD > LLLD, LDLL > LLDL). cyclo(-L-Asu(NHOH)-D-Tyr(Me)-L-Ile-D-Pro-) or CHAP31 was selected as one of the strongest CHAPs, and its biological activity was characterized further. CHAP31 was much more stable in the presence of cultured cells (t(1/2) > 3000 h) than trichostatin A (t(1/2) = 14.7 h) or trapoxin A (t(1/2) = 2.10 h). CHAP31 exhibited antitumor activity in C57BL x DBA/2 F(1) (BD2F(1)) mice bearing B16/BL6 tumor cells. Furthermore, CHAP31 inhibited the growth in four of five human tumor lines implanted into nude mice. These results suggest CHAP31 to be promising as a novel therapeutic agent for cancer treatment.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Péptidos Cíclicos/farmacología , Animales , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Humanos , Ácidos Hidroxámicos/química , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/enzimología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Desnudos , Péptidos Cíclicos/química , Prolina/química , Estereoisomerismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Platinum cytotoxics play an important role globally in the management of solid tumours. Cisplatin sets the standard for efficacy in both regions with careful administration to reduce nephrotoxicity. Carboplatin is associated with neurotoxicity, but has become the leading product in the US due largely to the easier to manage toxicity profile. Both agents have been widely used in both registered and non registered indications and are frequently combined with other cytotoxics. In Japan, cisplatin has been used successfully at low doses in combination with 5-FU based regimens and appears to achieve a synergistic effect, but controlled data are not yet available. More recently oxaliplatin (Europe) and nedaplatin (in Japan) have been introduced, but their clinical roles in therapy have yet to be established. One of the limiting features of the first generation of platinum compounds is that a significant proportion of tumours develop cross resistance to platins due to either changes in uptake or excretion, intracellular detoxification or accelerated DNA repair. The forum discussed the possibility for the development of better new platinum compounds, A new platin agent which had lower toxicity and higher efficacy across a wide range of cancers without the development of resistance would be a significant step forward. If the tolerability profile was suitable, an oral formulation may improve the quality of life for patients but this must not be at the expense of efficacy. Even after the introduction of new target based drugs, platinum cytotoxics are likely to be used to reduce the tumour mass and in some cases can be expected to potentiate the effects of the new agents. In preclinical studies, ZD0473 has been shown to by-pass some major mechanisms of resistance and has the potential to achieve these objectives and is now being evaluated in clinical studies in both Japan and the West.