RESUMO
In melanoma and other cancers, invasion, epithelial-to-mesenchymal transition, metastasis and cancer stem cell maintenance are regulated by transcription factors including the Snail family. Slug (Snail2) protein generally supports migration and apoptosis resistance. However, its role in melanoma is not completely understood. The present study investigated the transcriptional regulation of the SLUG gene in melanoma. It demonstrated that SLUG is under the control of the Hedgehog/GLI signaling pathway and is activated predominantly by the transcription factor GLI2. The SLUG gene promoter contains a high number of GLI-binding sites. Slug expression is activated by GLI factors in reporter assays and inhibited by GANT61 (GLI inhibitor) and cyclopamine (SMO inhibitor). SLUG mRNA levels are lowered by GANT61 as assessed by reverse transcription-quantitative PCR. Chromatin immunoprecipitation revealed abundant binding of factors GLI1-3 in the four subregions of the proximal SLUG promoter. Notably, melanoma-associated transcription factor (MITF) is an imperfect activator of the SLUG promoter in reporter assays, and downregulation of MITF had no effect on endogenous Slug protein levels. Immunohistochemical analysis confirmed the above findings and showed MITF-negative regions in metastatic melanoma that were positive for GLI2 and Slug. Taken together, the results demonstrated a previously unrecognized transcriptional activation mechanism of the SLUG gene, which may represent its main regulation of expression in melanoma cells.
Assuntos
Proteínas Hedgehog , Melanoma , Humanos , Proteínas Hedgehog/genética , Melanoma/genética , Fatores de Transcrição/genética , Apoptose , Transdução de SinaisRESUMO
Celiac disease (CD) is a disorder that affects both children and adults. Over the few last decades, several new atypical cases have been identified through improved diagnostic tools. On the other hand, the onset of CD at a later age, including atypical CD forms whose clinical picture overlaps with other autoimmune diseases, shows that currently there are several unknown gene mutations, which could be responsible for the disease development. Non-celiac gluten sensitivity (NCGS) is entity included by the ingestion of gluten leading to intestinal, or extraintestinal symptoms that improve once the gluten is removed from the nutrition. In this article relationships between genetically modified rodent animals with previously unknown multiple organ changes and CD, respectively NCGS are reviewed. Relationships between the small bowel histological changes and other organs pathology are discussed. Results of research document that changes have similar genetic background and can develop to serious autoimmune systematic diseases, including small bowel inflammation resembling atypical CD or NCGS. These may have extra-intestinal symptomatology but without a clear explanation of causes and differences in their manifestations. Research on animal models helps to discover links between several disorders associated with gastrointestinal damage. New methods based on individual gene mutations can help in atypical adult CD and NCGS recognitions in the future.
Assuntos
Doença Celíaca , Roedores , Animais , Doença Celíaca/genética , Glutens , Modelos AnimaisRESUMO
Metastasized malignant melanoma has a poor prognosis because of its intrinsic resistance to chemotherapy and radiotherapy. The central role in the melanoma transcriptional network has the transcription factor MITF (microphthalmia-associated transcription factor). It has been shown recently that the expression of MITF and some of its target genes require the SWI/SNF chromatin remodeling complex. Here we demonstrate that survival of melanoma cells requires functional SWI/SNF complex not only by supporting expression of MITF and its targets and but also by activating expression of prosurvival proteins not directly regulated by MITF. Microarray analysis revealed that besides the MITF-driven genes, expression of proteins like osteopontin, IGF1, TGFß2 and survivin, the factors known to be generally associated with progression of tumors and the antiapoptotic properties, were reduced in acute BRG1-depleted 501mel cells. Western blots and RT-PCR confirmed the microarray findings. These proteins have been verified to be expressed independently of MITF, because MITF depletion did not impair their expression. Because these genes are not regulated by MITF, the data suggests that loss of BRG1-based SWI/SNF complexes negatively affects survival pathways beyond the MITF cascade. Immunohistochemistry showed high expression of both BRM and BRG1 in primary melanomas. Exogenous CDK2, osteopontin, or IGF1 each alone partly relieved the block of proliferation imposed by BRG1 depletion, implicating that more factors, besides the MITF target genes, are involved in melanoma cell survival. Together these results demonstrate an essential role of SWI/SNF for the expression of MITF-dependent and MITF-independent prosurvival factors in melanoma cells and suggest that SWI/SNF may be a potential and effective target in melanoma therapy.
Assuntos
Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , DNA Helicases/metabolismo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Nevo/genética , Nevo/metabolismo , Nevo/patologia , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteopontina/genética , Osteopontina/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismoRESUMO
Comparison of HPLC methods using conventional particle-based and monolithic columns for determination of indomethacin and its two degradation products, viz. 5-methoxy-2-methylindoleacetic acid and 4-chlorobenzoic acid, was carried out. Ketoprofen was used as an internal standard for data evaluation throughout the study. Conventional separation was based on analytical column Zorbax SB-Phenyl (75x4.6 mm; 3.5 microm particles) used with a mobile phase composition of acetonitrile and phosphoric acid 0.2% (50:50, v/v) and isocratic flow at 0.6 mL/min. Three different lengths of Chromolith columns RP-18e (25x4.6 mm, 50x4.6 mm, and 100x3 mm) were tested with respect to the validation parameters peak asymmetry, resolution, height equivalent to a theoretical plate, repeatability, and after optimisation compared to values obtained using a conventional Zorbax column. The developed methods were used to determine all three compounds in a pharmaceutical formulation--Indobene gel. Chromatographic parameters were comparable to those of a conventional particle-based column. The analysis time was shortened as expected (retention times were lowered by a factor of two). Moreover, the repeatability of peak areas and retention times obtained with a 50 mm monolithic column was greatly improved (RSD values were lower than 0.40%).
