RESUMEN
BACKGROUND AND OBJECTIVE: Periodontal ligament stem cells (PDLSCs) from the periodontal ligament tissue were recently identified as mesenchymal stem cells (MSCs). The capabilities of PDLSCs in periodontal tissue or bone regeneration have been reported, but their immunomodulatory role in T-cell immune responses via dendritic cells (DCs), known as the most potent antigen-presenting cell, has not been studied. The aim of this study is to understand the immunological function of homogeneous human STRO-1+ CD146+ PDLSCs in DC-mediated T-cell immune responses to modulate the periodontal disease process. MATERIAL AND METHODS: We utilized highly purified (> 95%) human STRO-1+ CD146+ PDLSCs and human bone marrow mesenchymal stem cells (BMSCs). Each stem cell was co-cultured with human monocyte-derived DCs in the presence of lipopolysaccharide isolated from Porphyromonas gingivalis, a major pathogenic bacterium responsible for periodontal disease, in vitro to examine the immunological effect of each stem cell on DCs and DC-mediated T-cell proliferation. RESULTS: We discovered that STRO-1+ CD146+ PDLSCs, as well as BMSCs, significantly decreased the level of non-classical major histocompatibility complex glycoprotein CD1b on DCs, resulting in defective T-cell proliferation, whereas most human leukocyte antigens and the co-stimulatory molecules CD80 and CD86 in/on DCs were not significantly affected by the presence of BMSCs or STRO-1+ CD146+ PDLSCs. CONCLUSIONS: This study unveiled an immunomodulatory role of STRO-1+ CD146+ PDLSCs in negatively regulating DC-mediated T-cell immune responses, demonstrating their potential to be utilized in promising new stem cell therapies.
Asunto(s)
Antígenos CD1/metabolismo , Células Dendríticas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ligamento Periodontal/metabolismo , Linfocitos T/metabolismo , Técnicas de Cocultivo , Regulación hacia Abajo , Humanos , Activación de Linfocitos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
DNAJB9 is a recently isolated member of the molecular chaperone gene family, whose precise function is largely unknown. In the present study, we have identified DNAJB9 as an inducible gene of the tumor suppressor p53. DNAJB9 expression was induced by p53 or genotoxic stress in a p53-dependent manner, which was mediated by the Ras/Raf/ERK pathway. In addition, depletion of DNAJB9 by using siRNAs greatly increased genotoxic stress/p53-induced apoptosis, suggesting that DNAJB9 inhibits the pro-apoptotic function of p53. We also found that DNAJB9 physically interacts with p53 through its J domain, through which it inhibits the pro-apoptotic function of p53. Moreover, DNAJB9 colocalized with p53 in both cytoplasm and nucleus under genotoxic conditions. Together, these results demonstrate that DNAJB9 is a downstream target of p53 that belongs to the group of negative feedback regulators of p53.
Asunto(s)
Apoptosis , Daño del ADN , Proteínas del Choque Térmico HSP40/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas del Choque Térmico HSP40/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We aim to study the therapeutic effects of HBsAg-activated DCs and cytokine-induced killer (CIK) cells as adoptive immunotherapy in patients with Chronic Hepatitis B (CHB). Autologous HBsAg-activated DC-CIK cells were infused into patients with CHB to evaluate their effect on HBV-DNA, HBsAg, ALT, etc. The viral load in the treatment group decreased significantly (P < 0.001), while that in the control group did not decrease (P > 0.05). Twenty-one patients (63.6% efficiency) in the treatment group had a viral response (≥2 log decrease in viral load), while four patients (14.8% efficiency) from the control group had a viral response. There were significant differences in the viral responses of the two groups (the control group 63.6% versus the control group 14.8%, P < 0.001). We concluded that the immunity was enhanced after HBsAg activation in DCs and CIK cells. Reinfusion of autologous HBsAg-activated DC-CIK cells inhibited HBV proliferation in 21 of 33 (63.6%) patients.
Asunto(s)
Células Asesinas Inducidas por Citocinas/inmunología , Células Dendríticas/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Adolescente , Traslado Adoptivo/métodos , Adulto , Células Cultivadas , Células Asesinas Inducidas por Citocinas/trasplante , Células Dendríticas/trasplante , Femenino , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/terapia , Hepatitis B Crónica/virología , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Resultado del Tratamiento , Carga Viral/inmunología , Replicación Viral/inmunología , Adulto JovenRESUMEN
This study was conducted to identify proteins associated with the endogenous synthesis of conjugated linoleic acid (CLA) from trans-vaccenic acid (TVA; trans-11 C18:1, a precursor for CLA endogenous synthesis) in mammary tissues. Six lactating goats were divided into 2 groups. One group was given an intravenous bolus injection of TVA (150mg) twice daily over 4 d; the other group received saline injections. Treatment with TVA increased the concentration of cis-9,trans-11 CLA and TVA in goat milk. Additionally, TVA treatment increased the expression of stearoyl-CoA desaturase (SCD) in mammary tissue. Using 2-dimensional gel electrophoresis and electrospray ionization quadrupole time-of-flight mass spectrometry, 3 proteins affected by infusions of TVA were identified. Proteasome (prosome, macropain) subunit α type 5 (PSMA5) was upregulated, whereas peroxiredoxin-1 and translationally controlled tumor protein 1 were downregulated in TVA-treated animals compared with the vehicle-injected controls. Only the effect of TVA on PSMA5 could be confirmed by Western blot analysis. To further explore the regulation of PSMA5 in mammary epithelial cells when TVA is converted into CLA, we used a differentiated bovine mammary epithelial cell line treated with TVA for 6h. Changes in cis-9,trans-11 CLA concentrations and mRNA expression patterns of both SCD and PSMA5 were monitored. The concentration of cis-9,trans-11 CLA increased after TVA treatment. The mRNA expression level of PSMA5 was significantly elevated to 6h, but SCD mRNA expression only increased in 2h after TVA treatment. These results indicate that PSMA5 is highly expressed in goat mammary tissue and bovine mammary epithelial cells when TVA is converted into CLA. Our data suggest that PSMA5 protein is associated with CLA biosynthesis in mammary tissue.
Asunto(s)
Cabras/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Glándulas Mamarias Animales/metabolismo , Leche/química , Ácidos Oléicos/farmacología , Animales , Western Blotting , Bovinos , Línea Celular , Electroforesis en Gel Bidimensional/veterinaria , Femenino , Ácidos Linoleicos Conjugados/análisis , Ácidos Linoleicos Conjugados/genética , ARN/química , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Ionización de Electrospray/veterinariaRESUMEN
Peripheral nerve injury often results in neuropathic pain that is manifested as hyperalgesia, and allodynia. Several studies suggest a functional role for neuronal nitric oxide synthase (nNOS) in the development or maintenance of neuropathic pain, but such a contribution remains unclear. In our current study, we found that intraplantar injection of the NOS substrate L-arginine or NO donor 3-morpholino-synonimine (SIN-1) produced mechanical hypersensitivity that lasted more than 24 h. Following L5 spinal nerve ligation (L5 SNL), immunoreactivity for nNOS in the ipsilateral L5 but not L4 dorsal root ganglion (DRG) was dramatically increased in mainly small- and medium-sized neurons and non-neuronal cells. L5 SNL caused increased nNOS immunoreactivity in the ipsilateral sciatic nerve, mainly in Schwann cells and the ipsilateral glabrous hind paw skin, mainly on the basement membrane. Furthermore, total nNOS protein and mRNA in the ipsilateral sciatic nerve and hind paw skin were markedly upregulated following nerve injury. Intraplantar injection of the NOS inhibitor 7-nitroindazole (7-NI) or the non-specific NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME) effectively suppressed SNL-induced mechanical allodynia. Collectively, these data suggest that in the periphery nNOS upregulation induced by peripheral nerve injury contributes to mechanical hypersensitivity during the maintenance phase of neuropathic pain. Blocking nNOS signaling in the periphery may thus be a novel therapeutic strategy for the treatment of neuropathic pain.
Asunto(s)
Hiperalgesia/metabolismo , Neuralgia/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Arginina/farmacología , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Masculino , Molsidomina/análogos & derivados , Molsidomina/farmacología , Neuralgia/fisiopatología , Óxido Nítrico Sintasa de Tipo I/genética , Umbral del Dolor/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología , Ratas , Ratas Sprague-Dawley , Células de Schwann/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVES: Multidisciplinary treatment approaches have been found to be effective for chronic pain patients although there are large individual differences in outcomes. To increase overall treatment effects, tools are needed to identify patients most likely to benefit from tailored, comprehensive modular treatment schemes. DESIGN: The present study evaluates the effects of a multidisciplinary pain treatment allocation protocol in chronic pain patients and seeks to identify cognitive-behavioural predictors of outcome. Pain intensity, functional disability, depression, and use of medication in an intervention group of 110 chronic pain patients were compared to the outcomes of a 110 strong control group. RESULTS: Paired pre- and post-treatment t tests showed that all primary outcomes had significantly decreased in the intervention group with ANCOVAs revealing a main group effect for post-treatment pain intensity levels and functional disability. Paired t tests demonstrated both variables to have significantly reduced after treatment relative to the levels reported by the control group. Predictor analyses further showed higher levels of acceptance to significantly predict larger reductions in pain intensity in the intervention but not in the control group. CONCLUSION: The tested multidisciplinary allocation scheme for out-patient treatment of chronic pain complaints was effective in reducing pain intensity and functional disability. Findings also showed that especially those patients that are able to accept their condition are likely to profit most from the treatment in terms of pain reduction.
Asunto(s)
Terapia Cognitivo-Conductual , Conducta Cooperativa , Comunicación Interdisciplinaria , Manejo del Dolor , Dolor/psicología , Grupo de Atención al Paciente , Psicoterapia de Grupo , Adaptación Psicológica , Adulto , Atención Ambulatoria , Enfermedad Crónica , Terapia Combinada , Atención Integral de Salud , Femenino , Desamparo Adquirido , Humanos , Masculino , Persona de Mediana Edad , Clínicas de Dolor , Dimensión del Dolor , Estimulación Eléctrica Transcutánea del Nervio , Resultado del TratamientoRESUMEN
UNLABELLED: Transcutaneous electrical nerve stimulation (TENS) is an easy to use non-invasive analgesic intervention applied for diverse pain states. However, effects in man are still inconclusive, especially for chronic pain. Therefore, to explore the factors predicting result of TENS treatment in chronic pain we conducted a prospective, randomized, placebo-controlled trial (n=163), comparing high frequency TENS (n=81) with sham TENS (n=82). Patients' satisfaction (willingness to continue treatment; yes or no) and pain intensity (VAS) were used as outcome measures. The origin of pain and cognitive coping strategies were evaluated as possible predictors for result of TENS treatment. RESULTS: Fifty-eight percent of the patients in the TENS group and 42.7% of the sham-TENS group were satisfied with treatment result (chi square=3.8, p=0.05). No differences were found for pain intensity. Patients diagnosed with osteoarthritis and related disorders (especially of the vertebral column) or peripheral neuropathic pain were less satisfied with high frequency TENS (OR=0.12 (95% CI 0.04-0.43) and 0.06 (95% CI 0.006-0.67), respectively). Injury of bone and soft tissue (especially postsurgical pain disorder) provided the best results. Treatment modality or interactions with treatment modality did not predict intensity of pain as a result of treatment. We conclude, that predicting the effect of high frequency TENS in chronic pain depends on the choice of outcome measure. Predicting patients' satisfaction with treatment result is related to the origin of pain. Predicting pain intensity reflects mechanisms of pain behavior and perceived control of pain, independent of treatment modality. Pain catastrophizing did not predict TENS treatment outcome.
Asunto(s)
Manejo del Dolor , Dolor/psicología , Estimulación Eléctrica Transcutánea del Nervio/métodos , Adaptación Psicológica , Enfermedad Crónica , Método Doble Ciego , Humanos , Dolor/patología , Dimensión del Dolor/métodos , Satisfacción del Paciente , Valor Predictivo de las Pruebas , Estudios Prospectivos , Resultado del TratamientoRESUMEN
OBJECTIVE: Based on the fear-avoidance and helplessness models, the relative contribution of fear of pain, avoidance behavior, worrying, and helplessness were examined in relation to fluctuations in functional disability in chronic-pain patients. METHODS: A cohort of 181 chronic-pain patients first completed various questionnaires and kept a 7-day pain journal during a standard 3-month waiting-list period prior to their scheduled treatment at an Interdisciplinary Pain Centre and did so again immediately preceding the intervention. RESULTS: At baseline, fear of pain, avoidance behavior, and helplessness all predicted functional disability after 3 months. Stepwise regression analyses showed avoidance behavior to be the strongest predictor of change in functional disability followed by helplessness, thus both ahead of fear of pain. CONCLUSION: The current findings support the roles of both fear-avoidance factors and helplessness in the functional disability in chronic-pain patients awaiting treatment but revealed a central role for avoidance behavior.
Asunto(s)
Adaptación Psicológica , Ansiedad/psicología , Reacción de Prevención , Miedo/psicología , Desamparo Adquirido , Dolor/psicología , Adolescente , Adulto , Anciano , Ansiedad/etiología , Enfermedad Crónica , Estudios de Cohortes , Evaluación de la Discapacidad , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Dolor/complicaciones , Análisis de Regresión , Estadísticas no ParamétricasRESUMEN
OBJECTIVES: The goal of this study was to examine the relative contribution of helplessness, fear of pain, and passive pain-coping to pain level, disability, and depression in chronic pain patients attending an interdisciplinary pain center. METHODS: One hundred sixty-nine chronic pain patients who had entered treatment at an interdisciplinary pain center completed various questionnaires and a pain diary. RESULTS: Helplessness, fear of pain, and passive pain-coping strategies were all related to the pain level, disability, and depression. When comparing the contribution of the predictors in multiple regression analyses, helplessness was the only significant predictor for pain level. Helplessness and the passive behavioral pain-coping strategies of resting significantly predicted disability. The passive cognitive pain-coping strategy of worrying significantly predicted depression. CONCLUSIONS: These findings indicate a role for helplessness and passive pain-coping in chronic pain patients and suggest that both may be relevant in the treatment of pain level, disability, and/or depression.
Asunto(s)
Adaptación Psicológica , Trastornos de Ansiedad/epidemiología , Depresión/epidemiología , Evaluación de la Discapacidad , Desamparo Adquirido , Dolor/diagnóstico , Dolor/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Trastornos de Ansiedad/diagnóstico , Trastornos de Ansiedad/psicología , Enfermedad Crónica , Comorbilidad , Estudios Transversales , Depresión/diagnóstico , Depresión/psicología , Miedo/psicología , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Dolor/psicología , Clínicas de Dolor/estadística & datos numéricos , Manejo del Dolor , Dimensión del Dolor , Medición de Riesgo/métodos , Factores de Riesgo , Índice de Severidad de la Enfermedad , Encuestas y CuestionariosRESUMEN
Dexamethasone (DEX) pretreatment protected hepatocytes from TNF-alpha plus actinomycin D (ActD)-induced apoptosis by suppressing caspase-8 activation and the mitochondria-dependent apoptosis pathway. DEX treatment upregulated cellular FLICE inhibitory protein (cFLIP) expression, but did not alter the protein levels of Bcl-2, Bcl-xL, Mcl-1, and cIAP as well as Akt activation. The increased cFLIP mRNA level by DEX was inhibited by ActD, indicating that DEX upregulates cFLIP expression at the transcriptional step. DEX also inhibited Jo2-mediated hepatocyte apoptosis by blocking the formation of the death-inducing signaling complex and caspase-8 activation. Specific downregulation of cFLIP expression using siRNA reversed the antiapoptotic effect of DEX by increasing caspase-8 activation. Moreover, DEX administration into mice increased cFLIP expression in the liver and prevented Jo2-induced hepatic injury by inhibiting caspase-8 and -3 activities. Our results indicate that DEX exerts a protective role in death receptor-induced in vitro and in vivo hepatocyte apoptosis by upregulating cFLIP expression.
Asunto(s)
Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Hepatocitos/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Caspasa 3 , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Dactinomicina/antagonistas & inhibidores , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Galactosamina/antagonistas & inhibidores , Hepatocitos/citología , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Activación Transcripcional , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Regulación hacia Arriba , Receptor fas/metabolismoRESUMEN
Serum contains a variety of biomolecules, which play an important role in cell proliferation and survival. We sought to identify the serum factor responsible for mitigating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and to investigate its molecular mechanism. TRAIL induced effective apoptosis without serum, whereas bovine serum decreased apoptosis by suppressing cytochrome c release and caspase activation. Indeed, albumin-bound lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) inhibited TRAIL-induced apoptosis by suppressing caspase activation and cytochrome c release. LPA increased phosphatidylinositol 3-kinase (PI3K)-dependent Akt activation, cellular FLICE-inhibitory protein (cFLIP) expression, and Bad phosphorylation, resulting in inhibition of caspase-8 activation and Bad translocation to mitochondria. The antiapoptotic effect of LPA was abrogated by PI3K inhibitor, transfection with dominant-negative Akt, and specific downregulation of cFLIP expression using siRNA and further increased by siRNA-mediated suppression of Bad expression. Moreover, sera from ovarian cancer patients showed more protective effect against TRAIL-induced apoptosis than those from healthy donors, and this protection was suppressed by PI3K inhibitor. Our results indicate that albumin-bound LPA and S1P prevent TRAIL-induced apoptosis by upregulation of cFLIP expression and in part by Bad phosphorylation, through the activation of PI3K/Akt pathway.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Resistencia a Antineoplásicos/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisofosfolípidos/sangre , Glicoproteínas de Membrana/metabolismo , Neoplasias Ováricas/sangre , Esfingosina/análogos & derivados , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Animales , Proteínas Reguladoras de la Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Bovinos , Línea Celular Tumoral , Células Clonales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Lisofosfolípidos/farmacología , Masculino , Glicoproteínas de Membrana/farmacología , Persona de Mediana Edad , Neoplasias Ováricas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Esfingosina/sangre , Esfingosina/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteína Letal Asociada a bclRESUMEN
Fluorine-18 fluorodeoxyglucose (FDG), the most widely used radiopharmaceutical in positron emission tomography (PET) for oncological purposes, is unsuitable for imaging of bladder cancer owing to high excretion into the urine. More specific PET radiopharmaceuticals which are not excreted into urine would be welcome. Carbon-11 labelled choline (CHOL) is a new radiopharmaceutical potentially useful for tumour imaging and is not excreted into the urine. We prospectively studied the visualisation of bladder cancer using CHOL PET. Eighteen patients with bladder cancer and five healthy volunteers were included. Bladder cancer was first diagnosed by transurethral resection or by biopsy of the tumour. Next, PET images were performed before surgical treatment by cystectomy. The histopathological findings after cystectomy were used as the gold standard. PET images were performed on either an ECAT 951/31 or an ECAT Exact HR+ system. Attenuation-corrected PET images were obtained after injection of 400 MBq CHOL. PET images were analysed by two independent physicians using visual analysis and calculation of the standardised uptake value (SUV). In the normal bladder wall, the uptake of CHOL was low, and the bladder margin was only outlined by minimal urinary radioactivity, if present. In ten patients the tumour was detected correctly by CHOL PET, with an SUV of 4.7+/-3.6 (mean+/-SD). One false positive CHOL PET scan was seen in a patient with an indwelling catheter for 2 weeks prior to the PET scan. In two patients, lymph node metastases were detected by CHOL PET. A micrometastasis <5 mm was not visualised with CHOL PET. In seven patients, no residual tumour was found after surgery. In six of seven patients CHOL PET imaging was negative. In situ carcinoma, dysplasia and a non-invasive urothelial tumour (pTa) remained undetected in three of these six patients. Minimal to no urinary tract radioactivity was seen in 22/23 subjects. Non-specific uptake of CHOL was observed in the small bowel, rectum and prostate gland. CHOL uptake in bladder cancer was avid, visualising the tumour in the virtual absence of urinary radioactivity. No uptake of CHOL was seen in pre-malignant lesions or in small non-invasive tumours. Our results warrant further research into the value of CHOL PET in the clinical management of patients with bladder cancer.
Asunto(s)
Radioisótopos de Carbono , Carcinosarcoma/diagnóstico por imagen , Colina , Tomografía Computarizada de Emisión/métodos , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Carcinosarcoma/patología , Carcinosarcoma/cirugía , Cistectomía , Reacciones Falso Positivas , Humanos , Persona de Mediana Edad , Neoplasia Residual , Neoplasias Glandulares y Epiteliales/diagnóstico por imagen , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Glandulares y Epiteliales/cirugía , Radiofármacos , Sensibilidad y Especificidad , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/cirugíaRESUMEN
Modification of transcription factors would result in significant changes in the expression of related genes. Recently, the presence of transglutaminase (TGase) has been reported in nuclei, the biological significances of which have attracted a great concern. In this study, we tested the possibility that nuclear TGase would crosslink and regulate the activity of a glutamine-rich transcription factor Sp1. The addition of purified guinea pig liver TGase increased the binding activity of Sp1 to the target DNA sequence by gel electrophoretic mobility shift assay. The activity of the human p21WAF1 promoter containing six Sp1 binding sites was increased by the cotransfection of the TGase 2 gene, and two Sp1 sites at -82 and -69, relative to the transcription start site, were essential for the increased activity in human renal embryonic 293T cells. The activity of a minimal promoter containing three consensus Sp1 binding sites was increased by co-transfection of human TGase 2 gene. The amount of Sp1 protein was increased dramatically in TGase 2-transfected 293T cells and the Sp1 protein itself from HeLa cell nuclear extracts was crosslinked readily by purified TGase at 37 degrees C in the presence of Ca2+. These results suggest that the nuclear TGase might modulate the activity of the Sp1 transcription factor probably via the posttranslational or transcriptional modification of the factor by TGase.
Asunto(s)
Proteínas de Unión al GTP/farmacología , Factor de Transcripción Sp1/metabolismo , Transglutaminasas/farmacología , Animales , Sitios de Unión/genética , Línea Celular , Núcleo Celular/enzimología , Reactivos de Enlaces Cruzados/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Dimerización , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Cobayas , Células HeLa , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Factor de Transcripción Sp1/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Transfección , Transglutaminasas/metabolismoRESUMEN
Hemoglobin (Hb) is a toxic molecule responsible for the extreme lethality associated with experimental Escherichia coli peritonitis, but the mechanism has yet to be elucidated. Hb, but not globin, showed toxic effects in a live E. coli model but not in a model using killed E. coli. Methemoglobin, hematin, and the well-known Fenton reagents iron and iron-EDTA demonstrated the same lethal effect in E. coli peritonitis as Hb, while the addition of the Fenton inhibitors desferrioxamine (DF) and diethylenetriamine pentaacetate removed most of the cytotoxic activity of iron. Administration of a combined dose of superoxide dismutase and catalase minimized the action of Hb and iron-EDTA, suggesting that both O(2)(.-) and H(2)O(2) are involved in the toxic action of Hb in this rat model. The combination of the antioxidative enzymes and DF further suppressed iron-mediated lethality. An electron spin resonance technique with the spin-trapping reagent 5, 5-dimethyl-1-pyroline-N-oxide (DMPO) showed O(2)(.-) generation in the peritoneal fluid of rats injected with E. coli alone or E. coli plus iron-DF, and (.)OH generation was detected in the peritoneal fluid of the rats injected with iron-EDTA. Hb did not show any spin adduct of oxygen radicals, suggesting that Hb produces non-spin-trapping radical ferryl ion, which decayed the spin adduct of DMPO. In the presence of Hb or iron-EDTA, O(2)(-)-generating activity and viability of phagocytes decreased, whereas lipid peroxidation of peritoneal phagocytes increased. Generation of oxygen radicals and lipid peroxidation did not differ in the live and dead bacterial models. Bacterial numbers in the peritoneal cavity and blood were markedly increased in the live bacterial model with Hb and iron-EDTA. The Fenton inhibitor iron-DF prevented the loss of phagocyte function, lipid peroxidation, and bacterial proliferation. These results led us to conclude that the lethal toxicity of Hb in bacterial peritonitis is associated with a Fenton-type reaction, the products of which decrease phagocyte viability, through the induction of lipid peroxidation, allowing bacterial proliferation and resulting in mortality.
Asunto(s)
Infecciones por Escherichia coli/metabolismo , Hemoglobinas/metabolismo , Hemoglobinas/toxicidad , Peritonitis/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Quelantes/farmacología , Óxidos N-Cíclicos/farmacología , Deferoxamina/farmacología , Modelos Animales de Enfermedad , Ácido Edético/farmacología , Hemina/farmacología , Peróxido de Hidrógeno/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacos , Ácido Pentético/farmacología , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/farmacología , Superóxidos/farmacologíaRESUMEN
Although nitric oxide (NO) induces neuronal cell death under some conditions, it also can prevent apoptosis resulting from growth factor withdrawal. We investigated the molecular mechanism by which NO protects undifferentiated and differentiated PC12 cells from trophic factor deprivation-induced apoptosis. PC12 cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and cytochrome c release after 24 hr of serum withdrawal. The apoptosis of PC12 cells was inhibited by the addition of NO-generating donor S-nitroso-N-acetylpenicillamine (SNAP) (5-100 microM) and the specific caspase-3-like protease inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-cho) but not the YVADase (or caspase-1-like protease) inhibitor N-acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-cho). SNAP and Ac-DEVD-cho prevented the increase in DEVDase (caspase-3-like protease) activity. The SNAP-mediated suppression of DEVDase activity was only minimally reversed by the incubation of cell lysate with dithiothreitol, indicating that NO did not S-nitrosylate caspase-3-like proteases in PC12 cells. Western blot analysis showed that NO inhibited the proteolytic activation of caspase-3. The cGMP analog 8-bromo-cGMP (8-Br-cGMP) blocked apoptotic cell death, caspase-3 activity and activation, and cytochrome c release. The soluble guanylyl cyclase inhibitor 1-H-oxodiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (CODQ) significantly attenuated NO-mediated, but not 8-Br-cGMP-dependent, inhibition of apoptotic cell death, PARP cleavage, cytochrome c release, and DEVDase activity. Furthermore, the protein kinase G inhibitor KT5823 reversed both SNAP- and 8-Br-cGMP-mediated anti-apoptotic events. All these apoptotic phenomena were also suppressed by NO production through neuronal NO synthase gene transfer into PC12 cells. Furthermore, similar findings were observed in differentiated PC12 cells stimulated to undergo apoptosis by NO donors and NGF deprivation. These findings indicate that NO protects against PC12 cell death by inhibiting the activation of caspase proteases through cGMP production and activation of protein kinase G.
Asunto(s)
Apoptosis/fisiología , Inhibidores de Caspasas , GMP Cíclico/metabolismo , Óxido Nítrico/fisiología , Animales , Caspasa 3 , Diferenciación Celular/fisiología , Medio de Cultivo Libre de Suero , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Grupo Citocromo c/metabolismo , Activación Enzimática , Técnicas de Transferencia de Gen , Factores de Crecimiento Nervioso/deficiencia , Células PC12 , Péptido Hidrolasas/metabolismo , Ratas , Transducción de Señal/fisiologíaRESUMEN
Increased expression of Transglutaminases 2 (TGase 2, TGase C) was observed in PC-14 human lung cancer cells in association with doxorubicin resistance and the reduction of the enzyme expression was correlated with the increasing cytotoxicity of the drug (Han and Park, 1999). Hydrogen peroxide was suggested to be a key mediator for doxorubicin-induced DNA fragmentation leading to apoptosis. A possible role of hydrogen peroxide as a putative mediator of TGase 2 expression in the doxorubicin sensitive PC-14 cells was examined. TGase 2 expression was increased in PC-14 cells treated with doxorubicin in a dose-dependent manner resulting in the concomitant increase of reactive oxygen species. The rise of TGase 2 expression by doxorubicin treatment was inhibited by N-acetylcysteine or glutathione treatment, while direct addition of hydrogen peroxide to PC-14 cells induced TGase 2 expression. These results suggest that generation of hydrogen peroxide induced by doxorubicin treatment is one of the key factors in an enhancement of TGase 2 expression in PC-14 cells.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/farmacología , Peróxido de Hidrógeno/metabolismo , Neoplasias Pulmonares/enzimología , Transglutaminasas/biosíntesis , Acetilcisteína/farmacología , Inducción Enzimática , Regulación Enzimológica de la Expresión Génica , Glutatión/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Recently a role for transglutaminase 2 (TGase 2) in the drug resistance of cancer cells has been suggested, although the mechanism is unclear. In the present study, we observed that doxorubicin-resistant PC-14/ADR cells showed a ten-fold higher level of TGase 2 expression than drug-sensitive PC-14 cells. PC-14/ADR cells exhibited the classical multidrug resistance (MDR) phenotype, which was cross-resistant to vincristine, but not to cisplatin. The stepwise induction of resistance to doxorubicin and vincristine in PC-14 cells was accompanied by a gradual increase of TGase 2 expression, but this expression was not increased with induction of cisplatin resistance. To confirm the role of TGase 2 protein in the acquisition of drug resistance in PC-14 cells, the TGase 2 expression in PC-14/ADR cells was reduced by stable transfection with the antisense or ribozyme construct. In the clones showing reduced expression of TGase 2, lactate dehydrogenase released from drug-treated cells was increased in the presence of either MDR-related drugs (doxorubicin and vincristine) or a non-MDR-related drug (cisplatin). These data suggest that TGase 2 can play a role in the acquisition of drug resistance in PC-14 cells through a general cellular defense system other than the MDR-related system.
Asunto(s)
Adenocarcinoma/enzimología , Antineoplásicos/farmacología , Neoplasias Pulmonares/enzimología , Transglutaminasas/metabolismo , Cisplatino/farmacología , Cartilla de ADN , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transglutaminasas/genética , Células Tumorales Cultivadas/efectos de los fármacos , Vincristina/farmacologíaRESUMEN
Crosslinking has been suggested as one of the mechanisms involved in the aging process. Among the various random or enzyme-mediated crosslinking reactions, transglutaminase (TGase)-catalyzed crosslinking activity has been proposed for its possible involvement in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. Moreover, recent findings of TGase C as a putative signal transducer and cell cycle regulator has renewed interest in the study of TGase C in relation to aging phenomena. The ubiquitous presence of TGase C compared to the organ-specific localization of other types of TGases has attracted special attention as a cellular aging device. In the present investigation for in vitro studies, we have compared the pattern of TGase C in young and old human red blood cells, separated by density differentiation, and in early and late-passage or hydrogen peroxide-treated human primary fibroblasts. For in vivo study, we monitored the age-dependent changes of TGase C in the liver and brain tissues of 4, 12, 18, and 24-month-old Sprague-Dawley rats. We obtained evidence that both the activity and protein levels of TGase C were high in old RBC and late-passage or hydrogen peroxide-treated fibroblasts. Similar findings were seen in liver and brain tissue such as age-dependent increases in TGase activity and protein level in an organ-specific pattern. These data suggest that TGase C might play an active role in the cellular process with age.
Asunto(s)
Envejecimiento/metabolismo , Transglutaminasas/metabolismo , Adulto , Animales , Apoptosis/fisiología , Encéfalo/enzimología , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , División Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Senescencia Celular/fisiología , Niño , Reactivos de Enlaces Cruzados/metabolismo , Envejecimiento Eritrocítico/fisiología , Eritrocitos/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Peróxido de Hidrógeno/farmacología , Hígado/enzimología , Masculino , Oxidantes/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Flavonoid glycosides were metabolized to phenolic acids via aglycones by human intestinal microflora producing alpha-rhamnosidase, exo-beta-glucosidase, endo-beta-glucosidase and/or beta-glucuronidase. Rutin, hesperidin, naringin and poncirin were transformed to their aglycones by the bacteria producing alpha-rhamnosidase and beta-glucosidase or endo-beta-glucosidase, and baicalin, puerarin and daidzin were transformed to their aglycones by the bacteria producing beta-glucuronidase, C-glycosidase and beta-glycosidase, respectively. Anti-platelet activity and cytotoxicity of the metabolites of flavonoid glycosides by human intestinal bacteria were more effective than those of the parental compounds. 3,4-Dihydroxyphenylacetic acid and 4-hydroxyl-phenylacetic acid were more effective than rutin and quercetin on anti-platelet aggregation activity. 2,4,6-Trihydroxybenzaldehyde, quercetin and ponciretin were more effective than rutin and ponciretin on the cytotoxicity for tumor cell lines. We insist that these flavonoid glycosides should be natural prodrugs.
Asunto(s)
Bacterias/metabolismo , Flavonoides/metabolismo , Flavonoides/farmacología , Intestinos/microbiología , Biotransformación , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Flavonoides/toxicidad , Glicósidos/metabolismo , Humanos , Fenoles/metabolismo , Fenoles/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Tripsina/farmacología , Células Tumorales CultivadasRESUMEN
In contrast to intrinsic drug resistance, induced multidrug resistance in gastric cancer cells has not been well studied. Therefore, two doxorubicin-resistant cell lines, (SNU-1DOX, SNU-16DOX), were derived in vitro from gastric carcinoma cell lines (SNU-1, SNU-16) respectively, and their characteristics were investigated. These resistances were not associated with overexpression of mdrl, multidrug resistance associated protein 1 (MRP1), pi or liver class of glutathione S transferase (GST pi, GSTL), heat shock protein 70 (HSP70), p53 or transglutaminase C (TGC). Levels of p21WAF1 RNA and topoisomerase II protein were decreased in the SNU-16DOX, but not in SNU-1DOX. However, the subsequent enzyme activity of topoisomerase II in SNU-16DOX was not decreased, but rather increased in SNU-16DOX. Furthermore, both resistant cell lines showed lower uptake and higher efflux of doxorubicin and induced cross-resistance to etoposide and vincristine in addition to doxorubicin, indicating a multi-drug resistance phenotype. In summary, we report two gastric carcinoma cell lines exhibiting induced multidrug resistance phenotype and suggest that mdrl, MRP1, GST, TGC, HSP70 and p53 do not play important roles in induced drug resistance in these cell lines. The role of changes in topoisomerase II activity and/or protein is still inconclusive, and p21WAF1 is associated with induced multidrug resistance in the SNU-16DOX gastric carcinoma cell line.