RESUMEN
A-kinase anchor protein 12 (AKAP12; also known as Gravin) functions as a tumor suppressor in several human primary cancers. However, the potential correlation between histone deacetylase 3 (HDAC3) and AKAP12 and the underlying mechanisms remain unclear. Thus, in this study, in an aim to shed light into this matter, the expression levels of HDAC3 and AKAP12 in 96 colorectal cancer (CRC) and adjacent non-cancerous tissues, as well as in SW480 cells were examined by immunohistochemical, RT-qPCR and western blot analyses. The effects of HDAC3 and AKAP12 on the proliferation, apoptosis and metastasis of CRC cells were examined by cell counting kit-8 (CCK-8) assay, colony formation assays, flow cytometry, cell cycle analysis and Transwell assays. The results revealed that the reduction or loss of AKAP12 expression was detected in 69 (71.8%) of the 96 tissue specimens, whereas HDAC3 was upregulated in 50 (52.1%) of the 96 tumor tissue specimens. AKAP12 expression was markedly increased upon treatment with the HDAC3 inhibitors, trichostatin A (TSA) and RGFP966, at both the mRNA and protein level. Mechanistically, the direct binding of HDAC3 within the intron-1 region of AKAP12 was identified to be indispensable for the inhibition of AKAP12 expression. Moreover, the proliferation, colony-forming ability, cell cycle progression and the migration of the CRC cells were found to be promoted in response to AKAP12 silencing or AKAP12/HDAC3 co-silencing, whereas transfection with si-HDAC3 yielded opposite effects. Apart from the elevated expression of the anti-apoptotic protein, Bcl-2, after AKAP12 knockdown, the increased activity of PI3K/AKT signaling was found to be indispensable for AKAP12-mediated colony formation and migration. On the whole, these findings indicate that AKAP12 may be a potential prognostic predictor and therapeutic target for the treatment of CRC in combination with HDAC3.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Histona Desacetilasas/metabolismo , Adulto , Anciano , Apoptosis/fisiología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/patología , Regulación hacia ArribaRESUMEN
A pair of chiral ruthenium(II) complexes, Λ- and Δ-[Ru(bpy)2(p-BEPIP)](ClO4)2 [Λ- and Δ-RM0627; bpy = 2,2-bipyridine; p-BEPIP = 2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline], were prepared using the Sonogashira coupling reaction under microwave irradiation. The study shows that Λ-RM0627 emitted strong phosphorescence in the range 500-700 nm with a maximum at 594 nm when excited at 365 nm (the Stokes shift is about 227 nm), which was mainly located in the cell nucleus with red phosphorescence. Further studies using real-time phosphorescence observation confirmed that Λ-RM0627 can be taken up quickly by MDA-MB-231 cells and enriched in the nucleus. The in vitro and in vivo toxicities of Λ-RM0627 were also evaluated, and it was found that Λ-RM0627 slightly inhibited the growth of MDA-MB-231 breast cancer cells and HaCaT normal human epidermal cells and had little influence on the development of Zebrafish embryos at low concentration. In conclusion, the levoisomer of chiral ruthenium complexes can act as a potential phosphorescent probe that targets nuclei of living cells with low toxicity.
Asunto(s)
Neoplasias de la Mama/patología , Núcleo Celular/ultraestructura , Complejos de Coordinación/química , Fenantrolinas/química , Compuestos de Rutenio/química , Animales , Línea Celular Tumoral , Humanos , Análisis Espectral/métodos , Estereoisomerismo , Pez Cebra/embriologíaRESUMEN
Two ruthenium(II) complexes, [Ru(L)2(p-tFMPIP)](ClO4)2 (L = bpy, 1; phen, 2; p-tFMPIP = 2-(4-(trifluoromethyphenyl)-1H-imidazo[4,5f][1,10] phenanthroline)), were prepared by microwave-assisted synthesis technology. The inhibitory activity evaluated by MTT assay shown that 2 can inhibit the growth of MDA-MB-231 cells with inhibitory activity (IC50) of 16.3 µM, which was related to the induction of apoptosis. Besides, 2 exhibit low toxicity against normal HAcat cells. The inhibitory growth activity of both complexes related to the induction of apoptosis was also confirmed. Furthermore, the studies on the interaction of both complexes with c-myc G4 DNA shown that 1 and 2 can stabilize the conformation of c-myc G4 DNA in groove binding mode, which has been rational explained by using DFT theoretical calculation methods. In a word, this type of ruthenium(II) complexes can act as potential apoptosis inducers with low toxicity in clinic by stabilizing c-myc G4 DNA.
Asunto(s)
Apoptosis/efectos de los fármacos , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , ADN/química , G-Cuádruplex/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Rutenio/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Complejos de Coordinación/síntesis química , Complejos de Coordinación/metabolismo , ADN/metabolismo , Humanos , Fenantrolinas/química , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
MicroRNA-155 (miR-155) is overexpressed in many human cancers; however, the function of miR-155 is largely unknown in esophageal squamous cell carcinoma (ESCC). In the present study, we found that miR-155 is dramatically increased in ESCC tissues compared with the paired adjacent normal tissues, which suggested that miR-155 acts as an oncogene in ESCC. We predicted that tumor protein p53-induced nuclear protein 1 (TP53INP1) is a candidate target gene of miR-155 given that miR-155 expression decreased mRNA and protein levels of TP53INP1 as determined by RT-PCR and Western blot analysis. In addition, miR-155 and TP53INP1 showed a negative relation in ESCC tissues. Dual luciferase-based reporter assay indicated direct regulation of TP53INP1 by miR-155. Furthermore, we demonstrated that RNA interference of TP53INP1 increased the proliferation and colonies formation of EC-1 cells. Up-regulation of TP53INP1 abrogated miR-155 induced growth in EC-1 cells and mutation of TP53INP1 in 3'-UTR restored the effects when co-transfected with miR-155. We also indicated that overexpression of miR-155 significantly promoted the proliferation of EC-1 cells in vitro and the development of tumors in nude mice. Taken together, our study reveals that miR-155 acts as an oncogene by targeting TP53INP1 in ESCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Proteínas Portadoras/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Choque Térmico/metabolismo , MicroARNs/metabolismo , Oncogenes , Regiones no Traducidas 3' , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/genética , Humanos , Masculino , Ratones , Ratones Desnudos , MicroARNs/genética , Persona de Mediana Edad , Interferencia de ARN , Factores de Tiempo , Transfección , Carga Tumoral , Regulación hacia ArribaRESUMEN
OBJECTIVE: To investigate the effects of Xiezhuo Chubi Decoction (XZCBD) on the microRNA expression patterns of kidney in mice with hyperuricemia. METHODS: Sixty Kunming male mice were randomly divided into the high-, medium-, and low-dose XZCBD groups, benzbromarone group, model group, and control group. Except the control group, all mice were established with yeast method combined with uricase inhibition method to build hyperuricemia model, and the corresponding drugs (37.5 g/kg, 18.75 g/kg, 9.375 g/kg, and 0.02 g/kg per day) were administrated on the 7th day. On the 22nd day, the blood uric acid concentration was detected, and microRNA with obvious changes in kidney was screened with qRT-PCR. RESULTS: The uric acid in the model group was higher than that in the control group, and the levels of the uric acid were reduced after being treated with XZCBD; the differences among groups were significant (P<0.05). Compared with the control group, 32 kinds of microRNA expression changes were detected on the 15th day after being treated with high-dose XZCBD by microRNA expression profile screening. Among them, miR-34a could inhibit the expression of human urate anion exthanger 1, and miR-146a might have inhibited the inflammatory reaction. CONCLUSION: XZCBD could significantly reduce the serum uric acid level; its effect on hyperuricemia might be through affecting microRNA expressions.
Asunto(s)
Medicamentos Herbarios Chinos/uso terapéutico , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/genética , Riñón/metabolismo , MicroARNs/genética , Animales , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hiperuricemia/sangre , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones , Datos de Secuencia Molecular , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido Úrico/sangreRESUMEN
The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.
Asunto(s)
Diferenciación Celular , Condrocitos/química , Condrogénesis , Células Madre Mesenquimatosas/química , Factores de Transcripción/genética , Animales , Proteína Morfogenética Ósea 2/metabolismo , Cartílago/metabolismo , Línea Celular , Condrocitos/citología , Condrocitos/metabolismo , Cromatografía Liquida , Regulación hacia Abajo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Internet , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Proteómica , ARN Mensajero/genética , Proteínas Recombinantes/genética , Espectrometría de Masas en TándemRESUMEN
AIM: To study the effects of IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) on NF-kappaB, p53 and their downstream target genes. The relationship of NF-kappaB, p53, and IkappaBalpha was further discussed. METHODS: pECFP-IkappaBalpha, pECFP-IkappaBalphaM (amino acides 1-317, Ser32, 36A), pECFP-IkappaBalpha243N (amino acides 1-243), pECFP-IkappaBalpha244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-alpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappaB, p53 and IkappaBalpha (IkappaBalphaM, IkappaBalpha243N, IkappaBalpha244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IkappaBalpha and its mutants on the transprition level of NF-kappaB, NF-kappaB downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments beta-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader. RESULTS: Cells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IkappaBalpha, CFP-IkappaBalphaM, and CFP-IkappaBalpha243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IkappaBalpha244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-alpha, p53 and Bax in CFP-IkappaBalpha transfected cells did not change significantly, while in YFP-p65/CFP-IkappaBalpha co-transfected cells, IkappaBalpha decreased the transcription of p65 downstream gene TNF-alpha (2.24+/-0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08+/-0.891) (P<0.05). Phosphorylation defective IkappaBalpha (IkappaBalphaM) decreased the transcription levels of all the four genes compared with the control (P<0.05). The N terminus of IkappaBalpha (IkappaBalpha243N) increased the transcription of NF-kappaB (1.84+/-0.176) and TNF-alpha (1.51+/-0.203) a little bit. However, the C terminus of IkappaBalpha (IkappaBalpha244C) increased the transcription of NF-kappaB, TNF-alpha, p53 and Bax significantly (8.29+/-1.662, 14.16+/-2.121, 10.2+/-0.621, 3.72+/-0.346) (P<0.05). The CCK-8 experiment also showed that IkappaBalpha244C and p53 synergistically mediate apoptosis. CONCLUSIONS: IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) have different effects on NF-kappaB and p53 signaling pathways, according to their different structures. IkappaBalphaM bounds with NF-kappaB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IkappaBalpha244C may be co-factor in inducing apoptosis. The C terminal of IkappaBalpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.
Asunto(s)
Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Mutación , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Colorantes Fluorescentes , Regulación de la Expresión Génica/genética , Humanos , Proteínas Luminiscentes , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Unión Proteica , Transducción de Señal/genética , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Fibroblast growth factor-2 (FGF-2) is a multifunctional polypeptide that affects many cellular functions and phenomena. The wild-type recombinant human fibroblast growth factor rhFGF-2(W) and the mutant C78SC96S rhFGF-2(M) were expressed in Escherichia coli and their products were purified. The results by the means of fluorescence spectroscopy and CD spectrums, suggested that due to its decreased hydrophobicity rhFGF-2 is not deposited as an inclusion body. The mitogenic activity of the expressed rhFGF-2(M) on 3T3 fibroblasts was shown to be 10-fold more than the expressed rhFGF-2(W) of which the biological activity was a little less than that of the standard rhbFGF(W), indicating that the increased biological activity was due to the change of its secondary structure, dimerization and affinity binding to FGF receptor (FGFR).
Asunto(s)
Sustitución de Aminoácidos , Proliferación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Mutación Puntual , Células 3T3 , Animales , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Ratones , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To study the relationship between aberrant FHIT transcripts and hepatocellular carcinoma (HCC). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and single strand conformational polymorphism (SSCP) assays were used to analyze the transcripts and mutations of FHIT gene in 24 matched tumorous tissues and para-tumorous tissues from patients with HCC and in 4 normal liver tissues. RESULTS: Aberrant FHIT transcripts were observed in 11 out of 24 (46%) tumorous tissues and in 2 (8%) of the matched para-tumorous tissues. CONCLUSION: FHIT aberrant transcripts may play an important role in the pathogenesis of hepatocellular carcinoma.
Asunto(s)
Ácido Anhídrido Hidrolasas , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Humanos , Mutación , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIM: To obtain high-level expression of nonfusion recombinant human basic fibroblast growth factor (rhbFGF). METHODS: hbFGF cDNA was prepared from the total RNA of embryonic brain tissue. As a template, the obtained gene was used to clone nonfusion rhbFGF. New primers were employed to alter the translation initiation region (TIR) and reduce the G+C content through nucleotide change. Using pET-3C as vector, the cloned rhbFGF was expressed in BL21 (DE3). RESULTS: rhbFGF was expressed in E coli up to 30 % of the total cellular protein. Cation exchange and heparin affinity chromatography were employed to purify the target protein from the supernatant of bacteria lysate. The bioactivity of the purified rhbFGF was identical with the standard bFGF. CONCLUSION: Modification of TIR is an effective means to increase nonfusion expression rate of recombinant proteins, such as rhbFGF, in E coli.