RESUMEN
PURPOSE: The purpose of this study was to investigate the influencing factors for chest CT hysteresis and severity of coronavirus disease 2019 (COVID-19). METHODS: The chest CT data of patients with confirmed COVID-19 in 4 hospitals were retrospectively analyzed. An independent assessment was performed by one clinician using the DEXIN FACT Workstation Analysis System, and the assessment results were reviewed by another clinician. Furthermore, the mean hysteresis time was calculated according to the median time from progression to the most serious situation to improve chest CT in patients after fever relief. The optimal scaling regression analysis was performed by including variables with statistical significance in univariate analysis. In addition, a multivariate regression model was established to investigate the relationship of the percentage of lesion/total lung volume with lymphocyte and other variables. RESULTS: In the included 166 patients with COVID-19, the average value of the most serious percentage of lesion/total lung volume was 6.62, of which 90 patients with fever had an average hysteresis time of 4.5 days after symptom relief, with a similar trend observed in those without fever. Multivariate analysis revealed that lymphocyte count in peripheral blood and transcutaneous oxygen saturation decreased with the increase of the percentage of lesion/total lung volume. CONCLUSION: There is a hysteresis effect in the improvement of chest CT image relative to fever relief in patients with COVID-19. The pulmonary lesions may be related to the severity as well as decreased lymphocyte count or percutaneous oxygen saturation.
Asunto(s)
COVID-19 , Tomografía Computarizada por Rayos X , COVID-19/diagnóstico por imagen , Humanos , Pulmón/diagnóstico por imagen , Pulmón/fisiopatología , Saturación de Oxígeno , Estudios Retrospectivos , SARS-CoV-2RESUMEN
BACKGROUND: Previous qualitative studies suggested that the false negative rate of the T cell spot test for tuberculosis infection (T-SPOT.TB) is associated with many risk factors in tuberculosis patients. However, more precise quantitative studies are lacking. The purpose of this study was to investigate the factors affecting quantified spot-forming cells (SFCs) to early secreted antigenic target 6 kDa (ESAT-6) or culture filtrate protein 10 kDa (CFP-10) in patients with active tuberculosis. METHODS: We retrospectively analyzed the data of 360 patients who met the inclusion criteria. Using the SFCs to ESAT-6 or CFP-10 levels as dependent variables, variables with statistical significance in the univariate analysis were subjected to optimal scaling regression analysis. The combination of ESAT-6 and CFP-10 (i.e., T-SPOT.TB) was analyzed by the exact logistic regression model. RESULTS: The results showed that the SFCs to ESAT-6 regression model had statistical significance (P < 0.001) and that previous treatment and CD4+ and platelet counts were its independent risk factors (all P < 0.05). Their importance levels were 0.095, 0.596 and 0.100, respectively, with a total of 0.791. The SFCs to CFP-10 regression model also had statistical significance (P < 0.001); platelet distribution width and alpha-2 globulin were its independent risk factors (all P < 0.05). Their importance levels were 0.287 and 0.247, respectively, with a total of 0.534. The quantification graph showed that quantified SFCs to ESAT-6 or CFP-10 grading had a linear correlation with risk factors. Albumin-globulin ratio, CD4+ and CD8+ were independent risk factors for false negative T-SPOT.TB (all P < 0.05). CONCLUSIONS: In T-SPOT.TB-assisted diagnosis of patients with active tuberculosis, previous treatment, decreased CD4+ and platelet count might lead to the decreased SFCs to ESAT-6, decreased alpha-2 globulin and high platelet distribution width might lead to the decreased SFCs to CFP-10, decreased albumin-globulin ratio, CD4+ and CD8+ might lead to an increase in the false negative rate of the T-SPOT.TB.
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Antígenos Bacterianos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/diagnóstico , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Estudios Retrospectivos , Linfocitos T/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiología , Tuberculosis/patologíaRESUMEN
Visual working memory (VWM) is a cognitive memory buffer for temporarily processing and storing visual information. Previous studies suggest that its capacity is severely limited, and there is an ongoing debate on whether the storage capacity is object-based or feature-based in VWM. In this study, a change-detection task was employed to investigate whether and how task difficulty can affect VWM, specifically, its capacity and the unit of storage. Task difficulty was manipulated through the set size of memory items, memory fidelity required by the resolution of representation and the type of feature tested. We examined two types of stimuli: the single-feature type, where each memory item was composed of a single feature (color or shape), and the conjunctive-feature type, where each item was composed of a conjunction of two features (colored shape). Experiment 1 replicated the previous findings that memory capacity for colors was larger than shapes, and decreased with the resolution demand regardless of the type of stimuli. In Experiment 2, we analyzed and compared the results from single-feature items and conjunctive-feature items in the low- and high-resolution conditions while controlling for the number of to-be-remembered features. By directly matching the estimated capacity based on an object-unit and a feature-unit with the theoretical prediction, the results showed that the unit storage in VWM tended to be feature-based with low task difficulty, and to be object-based with high task difficulty. This suggests that VWM is dynamic and flexible, dependent on the load of the current task.
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Percepción de Forma/fisiología , Memoria a Corto Plazo/fisiología , Recuerdo Mental/fisiología , Percepción Visual/fisiología , Adulto , Percepción de Color/fisiología , Humanos , Reconocimiento Visual de Modelos/fisiología , Adulto JovenAsunto(s)
Pruebas Diagnósticas de Rutina/métodos , Pruebas Hematológicas/métodos , Modelos Estadísticos , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/diagnóstico , Adulto , Anciano , China/epidemiología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Tuberculosis/epidemiología , Tuberculosis/microbiologíaRESUMEN
Visual working memory (VWM) is a cognitive memory buffer for temporarily holding, processing, and manipulating visual information. Previous studies have demonstrated mixed results of the effect of depth perception on VWM, with some showing a beneficial effect while others not. In this study, we employed an adapted change detection paradigm to investigate the effects of two depth cues, binocular disparity and relative size. The memory array consisted of a set of pseudo-randomly positioned colored items, and the task was to judge whether the test item was changed compared to the memory item after a retention interval. We found that presenting the items in stereoscopic depth alone hardly affected VWM performance. When combining the two coherent depth cues, a significant larger VWM capacity of the perceptually closer-in-depth items was observed than that of the farther items, but the capacity for the two-depth-planes condition was not significantly different from that for the one-plane condition. Conflicting the two depth cues resulted in cancelling the beneficial effect of presenting items at a closer depth plane. The results indicate that depth perception could affect VWM, and the visual system may have an advantage in maintaining closer-in-depth objects in working memory.
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Memoria a Corto Plazo , Percepción Visual , Adulto , Humanos , Experimentación Humana no Terapéutica , Estimulación Luminosa/instrumentación , Estimulación Luminosa/métodosRESUMEN
It is known that the perceived size of an afterimage is modulated by the perceived distance between the observer and the depth plane on which the afterimage is projected (Emmert's law). Illusions like Ponzo demonstrate that illusory distance induced by depth cues can also affect the perceived size of an object. In this study, we report that the illusory distance not only modulates the perceived size of object's afterimage during the presence of the depth cues, but the modulation persists after the disappearance of the depth cues. We used an adapted version of the classic Ponzo illusion. Illusory depth perception was induced by linear perspective cues with two tilted lines converging at the upper boundary of the display. Two horizontal bars were placed between the two lines, resulting in a percept of the upper bar to be farther away than the lower bar. Observers were instructed to make judgment about the relative size of the afterimage of the lower and the upper bars after adaptation. When the perspective cues and the bars were static, the illusory effect of the Ponzo afterimage is consistent with that of the traditional size-distance illusion. When the perspective cues were flickering and the bars were static, only the afterimage of the latter was perceived, yet still a considerable amount of the illusory effect was perceived. The results could not be explained by memory of a prejudgment of the bar length during the adaptation phase. The findings suggest that cooccurrences of depth cues and object may link a depth marker for the object, so that the perceived size of the object or its afterimage is modulated by feedback of depth information from higher-level visual cortex even when there is no depth cues directly available on the retinal level.
Asunto(s)
Postimagen/fisiología , Señales (Psicología) , Percepción de Profundidad/fisiología , Percepción de Distancia/fisiología , Femenino , Humanos , Masculino , Ilusiones Ópticas/fisiología , Percepción del Tamaño/fisiologíaRESUMEN
BACKGROUND: Recent achievement in genetics and epigenetics has led to the exploration of the pathogenesis of systemic lupus erythematosus (SLE). Identification of differentially expressed genes and their regulatory mechanism(s) at whole-genome level will provide a comprehensive understanding of the development of SLE and its devastating complications, lupus nephritis (LN). METHODS: We performed whole-genome transcription and DNA methylation analysis in PBMC of 30 SLE patients, including 15 with LN (SLE LN(+)) and 15 without LN (SLE LN(-)), and 25 normal controls (NC) using HumanHT-12 Beadchips and Illumina Human Methy450 chips. The serum proinflammatory cytokines were quantified using Bio-plex Human Cytokine 27-plex assay. Differentially expressed genes and differentially methylated CpG were analyzed with GenomeStudio, R, and SAM software. The association between DNA methylation and gene expression were tested. Gene interaction pathways of the differentially expressed genes were analyzed by IPA software. RESULTS: We identified 552 upregulated genes and 550 downregulated genes in PBMC of SLE. Integration of DNA methylation and gene expression profiling showed that 334 upregulated genes were hypomethylated, and 479 downregulated genes were hypermethylated. Pathway analysis on the differential genes in SLE revealed significant enrichment in interferon (IFN) signaling and toll-like receptor (TLR) signaling pathways. Nine IFN- and seven TLR-related genes were identified and displayed step-wise increase in SLE LN(-) and SLE LN(+). Hypomethylated CpG sites were detected on these genes. The gene expressions for MX1, GPR84, and E2F2 were increased in SLE LN(+) as compared to SLE LN(-) patients. The serum levels of inflammatory cytokines, including IL17A, IP-10, bFGF, TNF-α, IL-6, IL-15, GM-CSF, IL-1RA, IL-5, and IL-12p70, were significantly elevated in SLE compared with NC. The levels of IL-15 and IL1RA correlated with their mRNA expression. The upregulation of IL-15 may be regulated by hypomethylated CpG sites in the promotor region of the gene. CONCLUSIONS: Our study has demonstrated that significant number of differential genes in SLE were involved in IFN, TLR signaling pathways, and inflammatory cytokines. The enrichment of differential genes has been associated with aberrant DNA methylation, which may be relevant to the pathogenesis of SLE. Our observations have laid the groundwork for further diagnostic and mechanistic studies of SLE and LN.
Asunto(s)
Metilación de ADN/genética , Regulación de la Expresión Génica/inmunología , Leucocitos Mononucleares , Lupus Eritematoso Sistémico/genética , Adulto , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Lupus Eritematoso Sistémico/inmunología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
1,1-Bis(3'-indolyl)-1-(p-anisyl)methane (DIM-C-pPhOCH3) activates the orphan receptor nerve growth factor-induced Balpha (Nur77) in cancer cells, and in this study, DIM-C-pPhOCH3 decreased Panc1 pancreatic cancer cell survival and arrested cells in G0-G1. These responses were accompanied by induction of the cyclin-dependent kinase inhibitor p21 in pancreatic cancer cells. Mechanistic studies showed that induction of p21 mRNA and protein by DIM-C-pPhOCH3 was Nur77 dependent but did not depend on Krüppel-like factor 4, which was also induced by DIM-C-pPhOCH3. Activation of p21 promoter constructs by DIM-C-pPhOCH3 required the GC-rich proximal region of the promoter, and results of RNA interference studies showed that Nur77-dependent activation of the p21 promoter involved interactions with Sp1 and Sp4 but not Sp3. Interactions of Nur77 with the p21 promoter in Panc1 cells treated with DIM-C-pPhOCH3 were also confirmed in chromatin immunoprecipitation assays. These data show that activation of nuclear Nur77 results in a novel pathway for induction of p21, which is independent of Nur77 response elements but dependent on Sp proteins bound to the GC-rich proximal region of the p21 promoter.
Asunto(s)
Anisoles/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Indoles/farmacología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Apoptosis/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Interpretación Estadística de Datos , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/metabolismo , Sustancias Intercalantes/metabolismo , Factor 4 Similar a Kruppel , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/biosíntesis , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Regiones Promotoras GenéticasRESUMEN
Bis(3'-indolyl)methane (DIM) is a metabolite of the phytochemical indole-3-carbinol, and both compounds exhibit a broad spectrum of anticancer activities. We have developed a series of synthetic symmetrical ring-substituted DIM analogues, including 5,5'-dibromoDIM, which are more potent than DIM as inhibitors of cancer cell and tumor growth. In colon cancer cells, 5,5'-dibromoDIM decreased cell proliferation and inhibited G(0)-G(1)- to S-phase progression, and this was accompanied by induction of the cyclin-dependent kinase inhibitor p21 in HT-29 and RKO colon cancer cells. Mechanistic studies showed that induction of p21 in both RKO (p53 wild-type) and HT-29 (p53 mutant) cells by 5,5'-dibromoDIM was Krüppel-like factor 4 (KLF4) dependent, and induction of p53 in RKO cells was also KLF4 dependent. Analysis of the p21 promoter in p53-dependent RKO cells showed that 5,5'-dibromoDIM activated p21 gene expression through the proximal GC-rich sites 1 and 2, and chromatin immunoprecipitation assays showed that KLF4 and p53 bound to this region of the promoter, whereas in HT-29 cells unidentified upstream cis-elements were required for induction of p21. 5,5'-DibromoDIM (30 mg/kg/d) also inhibited tumor growth and induced p21 in athymic nude mice bearing RKO cells as xenografts, showing that ring-substituted DIM such as 5,5'-dibromoDIM represent a novel class of mechanism-based drugs for clinical treatment of colon cancer.
Asunto(s)
Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Indoles/farmacología , Factores de Transcripción de Tipo Kruppel/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Desnudos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and Nur77 and induce receptor-dependent and -independent apoptotic pathways in colon and other cancer cells. Structure-activity studies show that the p-bromo (DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation of Nur77 and PPARgamma, induce expression of CCAAT/enhancer-binding protein homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the position of the phenyl substituents (para >/= meta >/= ortho) and required a free indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose) polymerase (PARP) that is consistent with activation of the extrinsic pathway of apoptosis. These responses were associated with the activation of c-jun N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also observed in athymic nude mice bearing RKO cell xenografts and treated with 30 mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer cells and tumors is related to a novel ER stress-independent activation of JNK.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/enzimología , Activación Enzimática/efectos de los fármacos , Indoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/efectos de los fármacos , Animales , Antineoplásicos/química , Western Blotting , Línea Celular Tumoral , Colágeno Tipo XI/efectos de los fármacos , Colágeno Tipo XI/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Humanos , Inmunohistoquímica , Inmunoprecipitación , Indoles/química , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Desnudos , Relación Estructura-Actividad , Factor de Transcripción CHOP/efectos de los fármacos , Factor de Transcripción CHOP/metabolismo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.
Asunto(s)
Estradiol/farmacología , Receptor alfa de Estrógeno/fisiología , Factores de Transcripción Sp/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción Sp/genética , Factores de Transcripción Sp/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/fisiología , Factor de Transcripción Sp3/genética , Factor de Transcripción Sp3/metabolismo , Factor de Transcripción Sp3/fisiología , TransfecciónRESUMEN
1,1-Bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) is a methylene-substituted diindolylmethane (C-DIM) analog that activates the orphan receptor nerve growth factor-induced-Balpha (NGFI-Balpha, Nur77). RNA interference studies with small inhibitory RNA for Nur77 demonstrate that DIM-C-pPhOCH(3) induces Nur77-dependent and -independent apoptosis, and this study has focused on delineating the Nur77-independent proapoptotic pathways induced by the C-DIM analog. DIM-C-pPhOCH(3) induced caspase-dependent apoptosis in RKO colon cancer cells through decreased mitochondrial membrane potential which is accompanied by increased mitochondrial bax/bcl-2 ratios and release of cytochrome c into the cytosol. DIM-C-pPhOCH(3) also induced phosphatidylinositol-3-kinase-dependent activation of early growth response gene-1 which, in turn, induced expression of the proapoptotic nonsteroidal anti-inflammatory drug-activated gene-1 (NAG1) in RKO and SW480 colon cancer cells. Moreover, DIM-C-pPhOCH(3) also induced NAG-1 expression in colon tumors in athymic nude mice bearing RKO cells as xenografts. DIM-C-pPhOCH(3) also activated the extrinsic apoptosis pathway through increased phosphorylation of c-jun N-terminal kinase which, in turn, activated C/EBP homologous transcription factor (CHOP) and death receptor 5 (DR5). Thus, the effectiveness of DIM-C-pPhOCH(3) as a tumor growth inhibitor is through activation of Nur77-dependent and -independent pathways.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Proteínas de Unión al ADN/fisiología , Indoles/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Esteroides/fisiología , Factores de Transcripción/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Inhibidores de Crecimiento/farmacología , Humanos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
Deletion analysis of several 17beta-estradiol (E(2))-responsive genes have identified GC-rich sites that are associated with hormone-induced transactivation in MCF-7 breast cancer cells. However, the role of individual specificity proteins (Sps) in mediating hormone-induced gene expression has not been unequivocally determined. In transient transfection studies using E(2)-responsive GC-rich promoters from the E(2)F1, carbamoylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), and retinoic acid receptor alpha (RAR alpha) genes, RNA interference using small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) decreased both basal and E(2)-induced transactivation. The contributions of individual Sp proteins to basal and E(2)-induced activity were promoter dependent. iSp1, iSp3, and iSp4 also significantly inhibited hormonal induction of E(2)F1, CAD, and RAR alpha mRNA levels; however, the enhanced inhibitory effects of the latter two small inhibitory RNAs suggest that Sp3 and Sp4 play a major role in estrogen receptor alpha/Sp-mediated gene expression in MCF-7 cells.
Asunto(s)
Neoplasias de la Mama/genética , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factores de Transcripción Sp/fisiología , Animales , Aspartato Carbamoiltransferasa/genética , Neoplasias de la Mama/patología , Células COS , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Chlorocebus aethiops , Dihidroorotasa/genética , Factor de Transcripción E2F1/metabolismo , Receptor alfa de Estrógeno/metabolismo , Humanos , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Factores de Transcripción Sp/metabolismo , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Betulinic acid (BA) is a phytochemical triterpenoid acid from bark extracts and is cytotoxic to cancer cells and tumors. We modified the A-ring of BA to give a 2-cyano-1-en-3-one moiety and the effects of the 2-cyano-lup-1-en-3-oxo-20-oic acid (CN-BA), 2-cyano derivative of BA, and its methyl ester (CN-BA-Me) were investigated in colon and pancreatic cancer cells. Both CN-BA and CN-BA-Me were highly cytotoxic to Panc-28 pancreatic and SW480 colon cancer cells. CN-BA and CN-BA-Me also induced differentiation in 3T3-L1 adipocytes, which exhibited a characteristic fat droplet accumulation induced by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Based on these results, we investigated the activities of CN-BA and CN-BA-Me as PPARgamma agonists using several receptor-mediated responses including activation of transfected PPARgamma-responsive constructs, induction of p21 in Panc-28 cells and induction of caveolin-1 and Krüppel-like factor 4 in colon cancer cells. The results clearly demonstrated that both CN-BA and CN-BA-Me activated PPARgamma-dependent responses in colon (caveolin-1) and pancreatic (p21) cancer cells, whereas induction of KLF4 by these compounds in colon cancer cells was PPARgamma independent and also dependent on cell context. The PPARgamma agonist activities of CN-BA and CN-BA-Me were structure-, response/gene- and cell context-dependent suggesting that these compounds are a novel class of selective PPARgamma modulators with potential for clinical treatment of colon and pancreatic cancer.
Asunto(s)
Neoplasias del Colon/metabolismo , PPAR gamma/agonistas , Neoplasias Pancreáticas/metabolismo , Triterpenos/farmacología , Células 3T3-L1 , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Factor 4 Similar a Kruppel , Ratones , Neoplasias Pancreáticas/patologíaRESUMEN
The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERalpha crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1alpha or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFkappaB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.
Asunto(s)
Antimutagênicos/toxicidad , Neoplasias de la Mama/tratamiento farmacológico , Cobalto/toxicidad , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Northern Blotting , Neoplasias de la Mama/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cicloheximida/farmacología , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Citosol/efectos de los fármacos , Citosol/metabolismo , Antagonismo de Drogas , Contaminantes Ambientales/toxicidad , Inducción Enzimática/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Dibenzodioxinas Policloradas/toxicidad , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Vascular endothelial growth factor receptor-1 (VEGFR1) is expressed in cancer cell lines and tumors and, in pancreatic and colon cancer cells, activation of VEGFR1 is linked to increased tumor migration and invasiveness. Tolfenamic acid, a nonsteroidal anti-inflammatory drug, decreases Sp protein expression in Panc-1 and L3.6pl pancreatic cancer cells, and this was accompanied by decreased VEGFR1 protein and mRNA and decreased luciferase activity on cells transfected with constructs (pVEGFR1) containing VEGFR1 promoter inserts. Comparable results were obtained in pancreatic cancer cells transfected with small inhibitory RNAs for Sp1, Sp3, and Sp4 and all three proteins bound to GC-rich elements in the VEGFR1 promoter. These results show that VEGFR1 is regulated by Sp proteins and that treatment with tolfenamic acid decreases expression of this critical angiogenic factor. Moreover, in vitro studies in Panc-1 cells show that activation of VEGFR1 by VEGFB to increase mitogen-activated protein kinase 1/2 phosphorylation and cell migration on collagen-coated plates is also inhibited by tolfenamic acid. Thus, targeted degradation of Sp proteins is highly effective for inhibiting VEGFR1 and associated angiogenic responses in pancreatic cancer.
Asunto(s)
Neoplasias Pancreáticas/metabolismo , Factores de Transcripción Sp/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Línea Celular Tumoral , Movimiento Celular/fisiología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Invasividad Neoplásica , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosforilación , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Factor de Transcripción Sp4/metabolismo , Transfección , Factor B de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , ortoaminobenzoatos/farmacologíaRESUMEN
The aryl hydrocarbon receptor (AhR) binds with high affinity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatics, but also binds with lower affinity to structurally diverse exogenous and endogenous chemicals. One study reported that 3-methylcholanthrene (3MC) activated the estrogen receptor (ER) through the AhR, which acts as co-regulatory protein, whereas a recent report showed that 3MC directly bound and activated ERalpha. This study also shows that the AhR agonists benzo[a]pyrene, 3,3',4,4'-tetrachlorobiphenyl, chrysin, 6-methyl-1,3,8-trichlorodibenzofuran, and 3,3'-diindolylmethane also induce ERalpha-dependent transactivation. Moreover, in chromatin immunoprecipitation assays, these compounds induce binding of AhR and ERalpha to the CYP1A1 and pS2 gene promoters, which is consistent with their activities as both selective AhR modulators (SAhRMs) and selective ER modulators (SERMs).
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/efectos de los fármacos , Receptores de Hidrocarburo de Aril/agonistas , Benzo(a)pireno/farmacología , Benzofuranos/farmacología , Sitios de Unión , Línea Celular Tumoral , Citocromo P-450 CYP1A1/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Receptor alfa de Estrógeno/metabolismo , Flavonoides/farmacología , Humanos , Indoles/farmacología , Ligandos , Metilcolantreno/farmacología , Bifenilos Policlorados/farmacología , Dibenzodioxinas Policloradas/farmacología , Presenilina-2/efectos de los fármacos , Presenilina-2/genética , Presenilina-2/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Relación Estructura-ActividadRESUMEN
The trifunctional carbamoylphosphate synthetase/aspartate transcarbamyltransferase/dihydroorotase (CAD) gene is hormone responsive in MCF-7 and ZR-75 breast cancer cells, and this response is inhibited by the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Estrogen-dependent induction of CAD mRNA and reporter gene activity in cells transfected with constructs (pCAD) containing hormone-responsive GC-rich CAD promoter inserts involves estrogen receptor alpha (ERalpha)/Sp1 interactions with these proximal GC-rich motifs. TCDD also inhibits hormone-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD constructs. The mechanism of inhibitory AhR-ERalpha/Sp1 cross talk was further investigated by chromatin immunoprecipitation (ChIP), and the results show that ERalpha/Sp1 and the AhR are constitutively bound to the CAD gene promoter and only minor changes are observed after treatment with 17beta-estradiol, TCDD, or their combination. However, examination of interactions of these transcription factors by fluorescence resonance energy transfer shows that E2 enhances ERalpha-Sp1 interactions, whereas cotreatment with TCDD significantly decreases interaction of these proteins. These results suggest that inhibitory AhR-ERalpha/Sp1 cross talk is due, in part, to enhanced association of AhR and ERalpha (also determined by fluorescence resonance energy transfer), which coordinately dissociates ER and Sp1 and decreases ERalpha/Sp1-mediated transactivation, whereas remaining associated with the CAD promoter. This represents a novel interaction between two ligand activated receptors where one receptor inhibits activation of the second receptor.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Factor de Transcripción Sp1/metabolismo , Animales , Aspartato Carbamoiltransferasa/genética , Aspartato Carbamoiltransferasa/metabolismo , Neoplasias de la Mama/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Línea Celular , Chlorocebus aethiops , Citocromo P-450 CYP1A1/genética , Dihidroorotasa/genética , Dihidroorotasa/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Humanos , Ligandos , Mutación/genética , Dibenzodioxinas Policloradas/farmacología , Regiones Promotoras GenéticasRESUMEN
Vascular endothelial growth factor receptor-2 (VEGFR2/KDR) is an important mediator of angiogenesis, and VEGFR2 mRNA is expressed in several pancreatic cancer cell lines. Deletion analysis of the VEGFR2 promoter in Panc-1, AsPC-1, and MiaPaCa-2 pancreatic cancer cells shows that the proximal region of the promoter is primarily responsible for VEGFR2 expression, and two GC-rich sites at -58 and -44 are critical elements in all three cell lines. Panc-1, AsPC-1, and MiaPaCa-2 cells also express Sp1, Sp3, and Sp4 proteins which bind to the GC-rich region of the VEGFR2 promoter in electrophoretic mobility shift and chromatin immunoprecipitation assays. RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4 decreases VEGFR2 mRNA and reporter gene activity in transfection assays, confirming that VEGFR2 expression in pancreatic cancer cells is regulated by Sp proteins. These results suggest that VEGFR2 cannot only be targeted by receptor tyrosine kinase inhibitors but also by drugs that downregulate Sp proteins or block Sp-dependent transactivation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Factor de Transcripción Sp4/metabolismo , Factores de Transcripción/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Línea Celular Tumoral , Humanos , Neoplasias Pancreáticas/genéticaRESUMEN
Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.