RESUMEN
A novel Gram-stain-negative bacterium, designated strain GY 70310T, was isolated from the intestinal tract of Konosirus punctatus collected from Minjiang River, China. Cells of the strain were rod-shaped and motile with a single polar flagellum. The result of 16S rRNA gene sequence analyses showed that strain GY 70310T was moderately related to Crenobacter luteus YIM 78141T (94.7%), Paludibacterium paludis KBP-21T (93.8%) and Crenobacter cavernae K1W11S-77T (93.0%). The draft genome of strain GY 70310T consisted of 3.4 Mbp with DNA G+C content of 66.3 mol%, which possessed genes putatively encoding nitrate reductase, nitrite oxidoreductase and urease. The novel strain showed a whole genome average nucleotide identity (OrthoANI) value of 77.1% and a digital DNA-DNA hybridization (dDDH) value of 22.4% with Crenobacter luteus DSM 27258T, followed by Crenobacter cavernae K1W11S-77T with OrthoANI and dDDH values of 76.4% and 20.6%, respectively. The major fatty acids (>10%) were identified as summed feature 3 (C16:1ω6c and/or iso-C15:0 2-OH, C16:1ω7c), C16:0 and C18:1ω7c. The major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified lipid and one unidentified phospholipid. On the basis of phylogenetic analyses, genotypic and chemotaxonomic characteristics, strain GY 70310T represents a novel species of the genus Crenobacter, for which the name Crenobacter intestini sp. nov., is proposed. The type strain is GY 70310T (= CGMCC 1.16821T = KCTC 62945T = NBRC 113900T).
Asunto(s)
Ácidos Grasos , Fosfolípidos , Técnicas de Tipificación Bacteriana , Betaproteobacteria , China , ADN Bacteriano/genética , Neisseriaceae , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
A novel Gram-stain-positive, catalase- and oxidase-positive, endospore-forming bacterium, designated GY 10110T, was isolated from mangrove soil collected from Qinzhou, Guangxi province, China. Cells were aerobic, motile with peritrichous flagella and rod-shaped. The strain grew at 15-37 °C (optimum, 28 °C), at 0-3â%(w/v) NaCl (1â%) and at pH 6.0-9.0 (pH 7.0). The major fatty acids of strain GY 10110T were anteiso-C15â:â0, iso-C15â:â0 and iso-C16â:â0. The predominant menaquinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The polar lipid profile comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphoglycolipid, glycolipid, two unidentified aminophospholipids and three unidentified phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain GY 10110T was closely related to Falsibacillus pallidus CCTCC AB 207188T (98.0â% sequence similarity) and Bacillus oceanisediminis CGMCC 1.10115T (96.9â%), respectively. The G+C content of strain GY 10110T based on the whole genome sequence was 42.3 mol%. The novel strain showed an average nucleotide identity (ANI) value of 77.8â% and a digital DNA-DNA hybridization (dDDH) value of 15.6â% with Falsibacillus pallidus CCTCC AB 207188T based on draft genome sequences, followed by Bacillus oceanisediminis CGMCC 1.10115T with ANI and dDDH values of 75.2 and 12.8â%, respectively. The results of the polyphasic taxonomic study, including phenotypic, chemotaxonomic and phylogenetic analysis, showed that strain GY 10110T represents a novel species of the genus Falsibacillus, for which the name Falsibacillus albus sp. nov. is proposed. The type strain is GY 10110T (=CGMCC 1.13648T=NBRC 113502T).
Asunto(s)
Bacillaceae/clasificación , Filogenia , Rhizophoraceae/microbiología , Microbiología del Suelo , Bacillaceae/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Composición de Base , Pared Celular/química , China , ADN Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Grasos/química , Glucolípidos/química , Hibridación de Ácido Nucleico , Peptidoglicano/química , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
A novel Gram-negative bacterium, non-motile and short rod-shaped, designated strain GY511T, was isolated from the intestines of fish collected from Maowei Sea, China. Growth occurred at pH 6.0-9.0 (optimum 7.0), 4-37 °C (optimum 28 °C) and at 0-2.5% (w/v) NaCl (optimum 1.0%). The result of 16S rRNA gene sequence analysis showed that strain GY511T is closely related to O. oryzae NBRC 113109T (97.6%), O. konkukae DSM 105395T (97.4%), Ottowia beijingensis CGMCC 1.12324T (95.9%), Ottowia pentelensis DSM 21699T (95.2%) and Ottowia thiooxydans DSM 14619T (95.0%). The DNA-DNA hybridization values of strain GY511T with O. oryzae NBRC 113109T and O. konkukae DSM 105395T were 35.4 ± 3.1% and 26.3 ± 1.8%, respectively. The major fatty acids (> 10%) were identified as summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0 and summed feature 8 (C18:1ω7c and/or C18:1ω6c) and the major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine, two unidentified aminolipids and an unidentified phospholipid. The G+C content of the genomic DNA was 62.9 mol%. Thiosulfate could be utilized as co-substrate for aerobic growth and was oxidised to sulfate. On the basis of phenotypic, chemotaxonomic and molecular data, strain GY511T is considered to represent a novel species of the genus Ottowia, for which the name Ottowia flava sp. nov. is proposed. The type strain is GY511T (= NBRC 113500T = DSM 107425T = CGMCC 1.13650T).
Asunto(s)
Comamonadaceae/clasificación , Comamonadaceae/aislamiento & purificación , Peces/microbiología , Intestinos/microbiología , Aerobiosis , Animales , Organismos Acuáticos/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , China , Análisis por Conglomerados , Comamonadaceae/genética , Comamonadaceae/fisiología , Citosol/química , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Ácidos Grasos/análisis , Concentración de Iones de Hidrógeno , Hibridación de Ácido Nucleico , Fosfolípidos/análisis , Filogenia , Quinonas/análisis , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Cloruro de Sodio/metabolismo , TemperaturaRESUMEN
BACKGROUND & AIMS: Activation of WNT signaling promotes the invasive activities of several types of cancer cells, but it is not clear if it regulates the same processes in colorectal cancer (CRC) cells, or what mechanisms are involved. We studied the expression and function of OVOL2, a member of the Ovo family of conserved zinc-finger transcription factors regulated by the WNT signaling pathway, in intestinal tumors of mice and human beings. METHODS: We analyzed the expression of OVOL2 protein and messenger RNA in CRC cell lines and tissue arrays, as well as CRC samples from patients who underwent surgery at Xiamen University in China from 2009 to 2012; clinical information also was collected. CRC cell lines (SW620) were infected with lentivirus expressing OVOL2, analyzed in migration and invasion assays, and injected into nude mice to assess tumor growth and metastasis. Tandem affinity purification was used to purify the OVOL2-containing complex from CRC cells; the complex was analyzed by liquid chromatography, tandem mass spectrometry, and immunoprecipitation experiments. Gene promoter activities were measured in luciferase reporter assays. We analyzed mice with an intestine-specific disruption of Ovol2 (Ovol2(flox/+) transgenic mice), as well as Apc(min/+) mice; these mice were crossed and analyzed. RESULTS: Analysis of data from patients indicated that the levels of OVOL2 messenger RNA were significantly lower in colon carcinomas than adenomas, and decreased significantly as carcinomas progressed from grades 2 to 4. Immunohistochemical analysis of a tissue array of 275 CRC samples showed a negative association between tumor stage and OVOL2 level. Overexpression of OVOL2 in SW620 cells decreased their migration and invasion, reduced markers of the epithelial-to-mesenchymal transition, and suppressed their metastasis as xenograft tumors in nude mice; knockdown of OVOL2 caused LS174T cells to transition from epithelial to mesenchymal phenotypes. OVOL2 bound T-cell factor (TCF)4 and ß-catenin, facilitating recruitment of histone deacetylase 1 to the TCF4-ß-catenin complex; this inhibited expression of epithelial-to-mesenchymal transition-related genes regulated by WNT, such as SLUG, in CRC cell lines. OVOL2 was a downstream target of WNT signaling in LS174T and SW480 cells. The OVOL2 promoter was hypermethylated in late-stage CRC specimens from patients and in SW620 cells; hypermethylation resulted in OVOL2 down-regulation and an inability to inhibit WNT signaling. Disruption of Ovol2 in Apc(min/+) mice increased WNT activity in intestinal tissues and the formation of invasive intestinal tumors. CONCLUSIONS: OVOL2 is a colorectal tumor suppressor that blocks WNT signaling by facilitating the recruitment of histone deacetylase 1 to the TCF4-ß-catenin complex. Strategies to increase levels of OVOL2 might be developed to reduce colorectal tumor progression and metastasis.
Asunto(s)
Movimiento Celular , Neoplasias Colorrectales/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Células CACO-2 , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Genotipo , Células HCT116 , Células HEK293 , Histona Desacetilasa 1/metabolismo , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Invasividad Neoplásica , Metástasis de la Neoplasia , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Factores de Tiempo , Factor de Transcripción 4 , Factores de Transcripción/genética , Transfección , Carga Tumoral , beta Catenina/metabolismoRESUMEN
Wnt signalling through ß-catenin and the lymphoid-enhancing factor 1/T-cell factor (LEF1/TCF) family of transcription factors maintains stem cell properties in both normal and malignant tissues; however, the underlying molecular pathway involved in this process has not been completely defined. Using a microRNA microarray screening assay, we identified let-7 miRNAs as downstream targets of the Wnt-ß-catenin pathway. Expression studies indicated that the Wnt-ß-catenin pathway suppresses mature let-7 miRNAs but not the primary transcripts, which suggests a post-transcriptional regulation of repression. Furthermore, we identified Lin28, a negative let-7 biogenesis regulator, as a novel direct downstream target of the Wnt-ß-catenin pathway. Loss of function of Lin28 impairs Wnt-ß-catenin-pathway-mediated let-7 inhibition and breast cancer stem cell expansion; enforced expression of let-7 blocks the Wnt-ß-catenin pathway-stimulated breast cancer stem cell phenotype. Finally, we demonstrated that the Wnt-ß-catenin pathway induces Lin28 upregulation and let-7 downregulation in both cancer samples and mouse tumour models. Moreover, the delivery of a modified lin28 siRNA or a let-7a agomir into the premalignant mammary tissues of MMTV-wnt-1 mice resulted in a complete rescue of the stem cell phenotype driven by the Wnt-ß-catenin pathway. These findings highlight a pivotal role for Lin28/let-7 in Wnt-ß-catenin-pathway-mediated cellular phenotypes. Thus, the Wnt-ß-catenin pathway, Lin28 and let-7 miRNAs, three of the most crucial stem cell regulators, connect in one signal cascade.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones , Ratones Noqueados , MicroARNs/genética , Células Madre Neoplásicas/patología , Proteínas de Unión al ARN/genética , Activación Transcripcional , Proteína Wnt1/genética , beta Catenina/genéticaRESUMEN
Wnt-ß-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4α is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4α maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt-ß-catenin signaling and HNF4α in the EMT process of HCC cells. Our results indicated that HNF4α expression was negatively regulated during Wnt-ß-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4α was found to directly associate with TCF4 to compete with ß-catenin but facilitate transcription co-repressor activities, thus inhibiting expression of EMT-related Wnt-ß-catenin targets. Moreover, HNF4α may control the switch between the transcriptional and adhesion functions of ß-catenin. Overexpression of HNF4α was found to completely compromise the Wnt-ß-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt-ß-catenin signaling and HNF4α in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt-ß-catenin signaling and HNF4α expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt-ß-catenin signaling or restoring HNF4α expression to prevent EMT.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Hepáticas Experimentales/patología , Masculino , Unión Proteica , Ratas , Ratas Wistar , Factores de Transcripción de la Familia Snail , Factor de Transcripción 4 , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
A novel Gram-stain-positive actinobacterium, designated strain SCSIO 11529(T), was isolated from tissues of the stony coral Galaxea fascicularis, and characterized by using a polyphasic approach. The temperature range for growth was 22-50 °C (optimum 28-45 °C), the pH range for growth was 6.0-8.0 (optimum pH 7.0), and the NaCl concentration range for growth was 0-7% (w/v) NaCl. The polar lipid profile contained diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine and an unknown polar lipid. The predominant menaquinone was MK-9(H4). The major fatty acids (>10%) were iso-C16:0, iso-C17:1ω6c, iso-C16:1 H and C16:1ω7c/iso-C15:0 2-OH. The DNA G+C content of strain SCSIO 11529(T) was 70.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SCSIO 11529(T) belongs to the genus Prauserella, with the closest neighbours being Prauserella marina MS498(T) (97.0% 16S rRNA gene sequence similarity), Prauserella rugosa DSM 43194(T) (96.4%) and Prauserella flava YIM 90630(T) (95.9%). Based on the evidence of the present study, strain SCSIO 11529(T) is considered to represent a novel species of the genus Prauserella, for which the name Prauserella coralliicola sp. nov. is proposed. The type strain is SCSIO 11529(T) ( = DSM 45821(T) = NBRC 109418(T)).
Asunto(s)
Actinomycetales/clasificación , Antozoos/microbiología , Filogenia , Actinomycetales/genética , Actinomycetales/aislamiento & purificación , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , China , Arrecifes de Coral , ADN Bacteriano/genética , Ácidos Grasos/química , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
Adipocytes arising from mesenchymal stem cells (MSCs) requires MSC adipocyte commitment and differentiation of preadipocytes to mature adipocytes. Several signaling and some cytokines affect the adipogenesis of MSCs. This review focuses on the roles of TGF-ß/SMAD signaling in adipocyte commitment of MSCs. BMP4 and BMP7 signaling are sufficient to induce adipocyte lineage determination of MSCs. The roles of BMP2, TGF-ß, and myostatin signaling in this process are unclear. Other TGF-ß/SMAD signaling such as BMP3 and BMP6 signaling have almost no effect on commitment because of limited research available, while GDF11 signaling inhibits adipocyte commitment in human MSCs. In this review, we summarize the available information on TGF-ß/SMAD signaling regulation of MSCs in adipocyte commitment. Deeper study of this commitment mechanism will offer new approaches in treating obesity, diabetes mellitus, and obesity-related metabolism syndrome.
Asunto(s)
Adipocitos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Transformador beta/genética , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
The orphan receptor TR3 functions in the nucleus as a transcription factor to negatively or positively regulate gene expression. c-Jun N-terminal kinase (JNK) phosphorylation plays an important role in modulating the nuclear functions of TR3. Although TR3 is the phosphorylation target of JNK, the regulatory mechanism of JNK on TR3 functions remains to be elucidated. Here we showed that JNK activator anisomycin induced TR3 phosphorylation through JNK1 rather than p38 and ERK signals, which is mediated by its upstream factors MAPK kinase 4 and MAPK kinase 7. We also identified the exact phosphorylation site of JNK to be serine 95 at the N terminus of TR3, around which a classical JNK phosphorylation motif exists. Furthermore, we demonstrated that TR3 phosphorylation by JNK coincided with its ubiquitination and degradation, resulting in the loss of its mitogenic activity. Finally, we showed that JNK-induced phosphorylation blocked the DNA binding property of TR3 and hence diminished its transactivation activity. Taken together, our findings revealed a novel cross talk between TR3 and JNK signal pathway and shed light on the mechanism of JNK phosphorylation-dependent regulation on TR3 nuclear functions.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , División Celular/fisiología , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Riñón/citología , Luciferasas/genética , Mitógenos/genética , Mitógenos/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fosforilación , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Serina/metabolismoRESUMEN
Great progress has been achieved in the study of the role of TGF-ß signaling in triggering epithelial-mesenchymal transition (EMT) in a variety of cancers; however, the regulation of TGF-ß signaling during EMT in mammary tumor metastasis has not been completely defined. In the present study, we demonstrated that OVOL2, a zinc finger transcription factor, inhibits TGF-ß signaling-induced EMT in mouse and human mammary tumor cells, as well as in mouse tumor models. Data from the Oncomine databases indicated a strong negative relationship between OVOL2 expression and breast cancer progression. Moreover, our experiments revealed that OVOL2 inhibits TGF-ß signaling at multiple levels, including inhibiting Smad4 mRNA expression and inducing Smad7 mRNA expression, blocking the binding between Smad4 and target DNA, and interfering with complex formation between Smad4 and Smad2/3. These findings reveal a novel mechanism that controls the TGF-ß signaling output level in vitro and in vivo. The modulation of these molecular processes may represent a strategy for inhibiting breast cancer invasion by restoring OVOL2 expression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal , Transducción de Señal , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Biomarcadores , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Estimación de Kaplan-Meier , Ratones , Metástasis de la Neoplasia , Pronóstico , Unión Proteica , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
All-trans retinoic acid (ATRA) affects cell proliferation, differentiation and apoptosis through its receptors, RARs and RXRs. Besides these, other receptors such as orphan receptor TR3, are also involved in the regulatory process of ATRA. However, how different receptors function in response to ATRA is still largely unknown. In the present study, we found that formation of TR3/RXRalpha heterodimers in the nucleus and their subsequent translocation into the cytoplasm, in association with regulation of apoptosis-related proteins Bcl-2, Bcl-xl and Bax, was critical for apoptosis induction by ATRA in breast cancer cells MCF-7. When such translocation was blocked by Leptomycin B (LMB), ATRA-induced apoptosis was consequently abolished. However, in ATRA-induced gastric cancer cells MGC80-3, RXRalpha heterodimerised with RARalpha but not with TR3, and remained in the nucleus exerting its effect on cell cycle regulation. When transfected with antisense-RARalpha, MGC80-3 cells changed from ATRA-sensitive to ATRA-resistant and most cells were arrested in the S phase, implying the importance of RARalpha in cell cycle regulation. Furthermore, we demonstrated that the effects of ATRA depend on the relative levels of TR3, RARalpha and RXRalpha expression in cancer cells. In ATRA-induced MCF-7 cells, highly expressed TR3 favours the formation of TR3/RXRalpha and promotes the TR3/RXRalpha signalling pathway causing apoptosis; while in ATRA-induced MGC80-3 cells, high expression of RARalpha favours the formation of RARalpha/RXRalpha and promotes the RXRalpha/RARalpha signalling pathway in mediating cell cycle regulation. In conclusion, these results reveal the novel mechanism that cellular expression and location of protein is associated with diverse signalling transduction pathways and the resultant physiological process.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Neoplasias Gástricas/metabolismo , Tretinoina/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptor alfa de Ácido Retinoico , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/patologíaRESUMEN
AIM: To investigate and determine the mechanism and signal pathway of tetradecanoylphorbol-1, 3-acetate (TPA) in degradation of RXRalpha. METHODS: Gastric cancer cell line, BGC-823 was used in the experiments. The expression level of RXRalpha protein was detected by Western blot. Nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation. Localization and translocation of RXRalpha were observed under laser-scanning confocal microscope through labeling specific anti-RXRalpha antibody and corresponding immunofluorescent antibody as secondary antibody. Different inhibitors were used as required. RESULTS: In BGC-823 cells, RXRalpha was expressed in the nucleus. When cells were treated with TPA, expression of RXRalpha was repressed in a time-dependent and TPA-concentration-dependent manner. Meanwhile, translocation of RXRalpha from the nucleus to the cytoplasm occurred, also in a time-dependent manner. When cells were pre-incubated with proteasome inhibitor MG132 for 3 hrs, followed by TPA for another 12 hrs, TPA-induced RXRalpha degradation was inhibited. Further observation of RXRalpha translocation in the presence of MG132 showed that MG-132 could block TPA-induced RXRalpha redistribution. Conversely, when RXRalpha translocation was inhibited by LMB, an inhibitor for blocking protein export from the nucleus, TPA could not repress expression of RXRalpha. CONCLUSION: TPA could induce the degradation of RXRalpha protein in BGC-823 cells, and this degradation is time- and TPA-concentration-dependent. Furthermore, the degradation of RXRalpha by TPA is via a proteasome pathway and associated with RXRalphatranslocation from the nucleus to the cytoplasm.