11 |
Modelagem molecular, síntese e avaliação da atividade biológica de potenciais antineoplásicos com a proteína hnRNP K e culturas de células tumorais / Molecular modeling, synthesis and biological evaluation of potential antineoplastics with hnRNP K and tumoral cell linesSilva, Vinicius Barreto da 23 September 2011 (has links)
A proteína hnRNP K é conhecida por seu papel nos múltiplos processos que compõe a expressão gênica, incluindo funções nos estágios de splicing, transcrição e tradução, desempenhadas, principalmente, através da ligação de seus domínios KH a nucleotídeos. A ativação inadequada da hnRNP K tem relação direta com a gênese de alguns tipos de câncer, sobretudo de cabeça e pescoço, mama e colo-retal, evidenciando a mesma como um atrativo alvo molecular para o desenvolvimento de novos fármacos antineoplásicos. Com o auxílio de técnicas in silico, foram identificados dois compostos orgânicos, um derivado de benzimidazol e outro derivado de fenilbenzamida, capazes de impedir a ligação da proteína hnRNP K a oligonucleotídeos in vitro. Aliando as técnicas de docking, campos de interação e dinâmica molecular foi possível sugerir que tais compostos apresentam características estruturais que permitem a realização de interações na fenda de ligação do domínio KH3, principalmente com os resíduos R40 e R59, os quais são considerados chaves no reconhecimento molecular de nucleotídeos. Os derivados de benzimidazol e fenilbenzamida constituem novos compostos de partida na busca de novos antineoplásicos que tem como alvo a proteína hnRNP K. Do ponto de vista de metabolismo e toxicidade, o derivado de fenilbenzamida parece ser mais promissor quando se investiga uma molécula para aplicação terapêutica, uma vez que gerou poucos alertas críticos de toxicidade, ao contrario do derivado de benzimidazol, que apresenta maior potencial genotóxico. Apesar de se ligarem aos domínios KH da hnRNP K e impedir sua complexação a nucelotídeos, ensaios com culturas de células mostraram apenas tênue atividade antitumoral para tais compostos, com maior redução de viabilidade celular, ao redor de 18% a 8,4 M, exibida pelo derivado de fenilbenzamida. Seguindo o princípio do análogo ativo, simulações de triagem virtual na busca de análogos dos ligantes da hnRNP K revelaram 21 novos derivados de benzimidazol ou fenilbenzamida na base de dados EXPRESS-Pick, dos quais 5 foram testados in vitro com a proteína e 3 novos ligantes identificados. Com o intuito de otimizar tais derivados, foram sugeridos in silico substituintes (potenciais bioisósteros) para os anéis dioxopirrolidínicos dos ligantes já identificados, guiando, assim, a futura síntese de novas substâncias com potencial atividade antitumoral. Além disso, o trabalho foi complementado através da proposição de síntese de novos derivados benzoxazepínicos acoplados a purinas, os quais também tem aplicação como antineoplásicos, entretanto por mecanismos que não envolvem a hnRNP K. A grande limitação desses derivados é a presença de um grupo nitro aromático, o qual é reconhecido por sua toxicidade pronunciada. Com o intuito de otimizar tais derivados, foram sugeridos in silico potenciais bioisósteros capazes de substituir o grupo nitro e guiar a síntese e novos derivados com atividade antitumoral e toxicidade reduzida. / hnRNP K protein is known for its role in the multiple processes that compose gene expression, including functions during splicing, transcription and translation, developed, mainly, by the binding of nucleotides to KH domains. Inadequate activation of hnRNP K induces the development of some types of cancer, including head and neck, breast and colorectal. In this way, hnRNP K is an attractive molecular target for antineoplastic drug design. Using in silico strategies, we have identified two organic compounds, a benzimidazole and a phenylbenzamide derivatives, able to prevent the natural binding of nucleotides to hnRNP K in vitro. Applying docking, molecular interaction fields and molecular dynamics simulations it was possible to propose that such compounds present structural characteristics capable to support intermolecular interactions inside KH3 domain binding cleft, mainly with R40 and R59 residues, which are extremely important during molecular recognition of nucleotides by hnRNP K. The benzimidazole and phenylbenzamide derivatives identified are novel lead compounds that can guide the design of new antineoplastic drugs targeting hnRNP K. Considering metabolic and toxicity predictions, the phenylbenzamide seems to be more promising than the benzimidazole derivative as a drug, once the benzimidazole presents genotoxic potential. Although both derivatives prevent the binding of nucleotides to hnRNP K, biological assays with tongue cancer cell lines revealed only a mild antitumoral activity for such compounds. Higher level of cell viability reduction, 18% at 8.4 M, was observed for the phenylbenzamide derivative. Following the similarity principle, virtual screening simulations were made intending to find novel benzimidazole and phenylbenzamide derivatives inside EXPRESS-Pick database. The search revealed 21 compounds, 5 of which were tested in vitro with hnRNP K, where 3 of them were active. Intending to optimize benzimidazole and phenylbenzamide derivatives in order to design more potent chemical entities, we have suggested in silico substituents as potential bioisosteric groups of the dioxopyrrolidine rings of hnRNP K ligands, guiding the future synthesis of novel compounds with enhanced antitumoral activity. Moreover, complementary work proposition was performed through the synthesis of benzoxazepin-purines, which also present antitumoral activity but not through hnRNP K pathway. The major limitation of such derivatives is the presence of a nitro aromatic group, which can be very toxic. 20 potential bioisosteric groups were proposed as fragment candidates to replace the nitro one in order to design novel antitumoral derivatives with reduced toxic potential.
|
12 |
Estudo in vitro e in vivo de novos compostos: com alvo-específico (hnRNP K e SET) ou com ação na mitocôndria para uso como antitumoral em carcinoma oral ou como citoprotetor em célula não-tumoral / Studies in vitro and in vivo novel compounds: with target-specific (hnRNP K and SET) or the mitochondrion action for use as antitumor in oral carcinoma cell or as cytoprotection in non-tumorGoto, Renata Nishida 12 September 2017 (has links)
Os avanços na compreensão da biologia das neoplasias de cabeça e pescoço têm aberto novas direções na ciência. As pesquisas estão sendo direcionadas para o desenvolvimento de terapias com alvos moleculares específicos, os quais são úteis tanto na predição dos tratamentos, quanto na seleção de pacientes que podem responder a uma determinada terapia com base nas alterações moleculares dos tumores. As proteínas hnRNP K e SET, recentemente identificadas como superexpressas em câncer de cabeça e pescoço, representam um novo e atrativo alvo terapêutico para esse tipo de câncer. As mitocôndrias também tem sido objeto de estudo, pois participam nos processos de morte celular por apoptose, e estão envolvidas na sobrevivência celular. Neste estudo avaliamos os efeitos in vitro e in vivo em carcinoma oral e em célula não-tumoral, de novos compostos com alvo-específico (hnRNP K e SET) ou com ação na mitocôndria, para aplicação tanto como antitumoral, como citoprotetor. A citotoxicidade dos compostos foi avaliada pelo método de resazurina nas linhagens tumorais de carcinoma de células escamosas de cabeça e pescoço (HN13, HN12, HN6 e CAL27). Os compostos 11 e 17, alvos específicos da proteína hnRNP K, apresentaram baixa citotoxicidade; o peptídeo OP449, alvo específico da proteína SET, e o composto YV-241, com ação na mitocôndria, apresentaram alta citotoxicidade, com valores de IC50 5,11 e 7,77 ?M, respectivamente. OP449 alterou as proteínas reguladas por SET e reduziu a proliferação das células tumorais no modelo de xenoenxerto ortotópico em camundongo BALB/ c nude; os resultados, porém, não foram significativos. A associação de OP449 com FTY720 promoveu um efeito sinérgico significativo (CID<0,7) na linhagem celular HN12 e reduziu os tumores do xenoenxerto no dorso em camundongos BALB/c nude. O composto YV-241 alterou o potencial de membrana mitocondrial das células tumorais e aumentou o número de mitocôndrias, observado por microscopia eletrônica de transmissão e por microscopia confocal, reduziu proteínas envolvidas com vias de sinalização de sobrevivência, proliferação, ciclo celular e angiogênese, e induziu apoptose com o envolvimento da mitocôndria. Além disso, o composto reduziu os tumores do modelo de xenoenxerto. O possível efeito citoprotetor do composto JM-E-H foi observado na linhagem NOK-SI, por meio da regulação da via de sinalização de HIF-1?. Portanto, OP449 + FTY720 e o composto YV-241 apresentam potencial terapêutico contra carcinoma oral, e o composto JM-E-H, potencial efeito citoprotetor / Advances in understanding the biology of head and neck cancer have opened new directions in science. Research is being directed at the development of therapies with specific molecular targets that are useful in predicting treatments or in selecting patients who may respond to a particular molecular therapy based on molecular changes of the tumors. The hnRNP K and SET proteins, recently identified as overexpressed in head and neck cancer, represent a new and attractive therapeutic target for this type of cancer. Mitochondria have also been the object of study, since they participate in the processes of cell death by apoptosis, and are involved in cell survival. In this work we evaluated the in vitro and in vivo effects in oral carcinoma and non-tumor cell of new compounds with specific target (hnRNP K and SET) or with action in mitocondria, for application either as antitumor or cytoprotectant. The cytotoxicity of the compounds was evaluated by the resazurin method in head and neck squamous cell carcinoma cell lines (HN13, HN12, HN6 and CAL27). Compounds 11 and 17, specific targets of hnRNP K protein, showed low cytotoxicity; the peptide OP449, specific target of SET protein, and compound YV-241, acting on mitochondria, showed high cytotoxicity, with IC50 values of 5.11 and 7. 77?M, respectively. OP449 altered SET-regulated proteins and decreased proliferation of tumor cells in the orthotopic xenograft model in BALB/c nude mouse. The results, however, were not significant. The association of OP449 with FTY720 caused a significant synergistic effect (CID <0.7) on HN12 cell line, and decreased the xenograft tumors. The YV-241 compound altered mitochondrial membrane potential of tumor cells and increased the number of mitochondria, observed by transmission electron microscopy and by confocal microscopy, reduced proteins involved with signaling pathways for survival, proliferation, cell cycle and angiogénesis, and induced apoptosis with the involvement of mitochondria. In addition,the compound reduced tumors of the xenograft model. The possible cytoprotective effect of the compound JM-E-H was observed in the NOK-SI lineage through the regulation of HIF-1? signaling pathway. Therefore, OP449 + FTY720 and compound YV-241 show therapeutic potential against oral carcinoma, and the compound JM-E-H, potential cytoprotective effect
|
13 |
Acúmulo da ribonucleoproteína heterogênea nuclear K em câncer de cabeça e pescoço: estudos mitocondriais / Accumulation of heterogeneous nuclear ribonucleoprotein K in head and neck cancer: mitochondrial studiesGarcia, Cristiana Bernadelli 03 April 2014 (has links)
A ribonucleoproteína heterogênea nuclear K (hnRNP K) é uma proteína envolvida em processos de expressão gênica e tem sido proposta como ligante de RNAs mensageiros mitocondriais. Apesar de ser considerada um marcador de pior prognóstico no câncer de cabeça e pescoço, o papel da hnRNP K nesta doença ainda é pouco conhecido. O objetivo deste trabalho foi estudar o envolvimento da hnRNP K na mitocôndria com ênfase na bioenergética e na identificação de novos potenciais ligantes de hnRNP K. As linhagens celulares utilizadas foram de carcinoma de cabeça e pescoço (HN13 e CAL 27) com silenciamento de RNA para hnRNP K e células HEK293 com super-expressão de hnRNP K. O efeito do acúmulo celular da hnRNP K na cadeia transportadora de elétrons mitocondrial foi avaliado por meio da atividade dos complexos mitocondriais I, II e V em células HN13. A redução do nível de hnRNP K usando RNA de interferência promoveu uma diminuição da atividade dos complexos nas células HN13, indicando o envolvimento da proteína na eficiência do transporte de elétrons na cadeia respiratória mitocondrial. Células HEK293 com super-expressão da hnRNP K (HEK293/hnRNP K) e as linhagens HN13 e CAL 27 com silenciamento e redução estável de hnRNP K foram utilizadas para determinar o papel de hnRNP K no potencial de membrana mitocondrial, níveis de ATP, produção de lactato e consumo de oxigênio. Células HEK293/hnRNP K comparadas ao controle apresentaram maior nível de ATP, menor potencial de membrana mitocondrial, menor consumo de oxigênio e maior produção de lactato. As células HN13 com redução da hnRNP K apresentaram níveis mais baixos de ATP, com menor liberação de lactato para o meio extracelular e maior consumo de oxigênio. Esses resultados sugerem que o acúmulo da proteína hnRNP K tem ação importante na mitocôndria por alterar o metabolismo bioenergético celular de fosforilação oxidativa para glicólise anaeróbica. A estratégia de co-imunoprecipitação usando anticorpos para hnRNP K, digestão de proteínas com tripsina e cromatografia líquida acoplada a espectrômetria de massa foi usada para encontrar novos potenciais ligantes de hnRNP K. A análise dos dados com o software SEPro identificou 57 proteínas candidatas a ligantes da hnRNP K. Três proteínas foram validadas por co-IP e Western blotting: o fator de transcrição mitocondrial PTCD3, YB1 e PSF. Propomos que a hnRNP K apresenta função na energética mitocondrial, e provavelmente, a sua interação com PTCD3 participa desta função. / Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a protein involved in gene expression processes, which has been proposed to bind mitochondrial mRNAs. Despite it to be considered a prognostic marker in cancer, the hnRNPK role in this disease is unknown. We addressed the involvement of hnRNP K in mitochondria with emphasis on bioenergetics and identification of new potential ligands of hnRNP K. The cell lines used were from head and neck squamous cell carcinoma (HN13 and CAL 27) with RNA silencing for hnRNP K , and HEK293 cells with overexpression of hnRNP K. The effects of cellular accumulation of hnRNP K in mitochondrial electron chain carriers were assessed by the activity of mitochondrial complexes I, II and V in HN13 cells. Reduced levels of hnRNP K using RNA interference promoted a decrease in the activity of the complexes in HN13 cells, indicating the involvement of the protein in the efficiency of the electron transport in mitochondrial respiratory chain. HEK293 cells with overexpression of hnRNP K (HEK293/hnRNP K) and HN13 and CAL 27 cells with silencing and stable reduction of hnRNP K were used to determine the role of hnRNP K in mitochondrial membrane potential, ATP levels, lactate production and oxygen consumption. HEK293/hnRNP K, compared to control cells, showed higher levels of ATP, reduced mitochondrial membrane potential, lower oxygen consumption and higher production of lactate. HN13 cells with reduced hnRNP K had lower ATP levels, with lower release of lactate to the extracellular medium and higher oxygen consumption. These results suggest that accumulation of hnRNP K protein plays a role in mitochondria by changing the cellular energetic metabolism from oxidative phosphorylation to glycolysis. The strategy of co-immunoprecipitation using antibodies for hnRNP K, protein digestion with trypsin, and liquid chromatography, coupled to mass spectrometer, were used to search for new potential ligands of hnRNP K. Data analysis with software SEPro identified 57 candidate proteins binding to hnRNP K. Three proteins were validated by co-IP and Western blotting: the mitochondrial transcription factor PTCD3, YB1, and PSF. We propose that hnRNP K plays a role in the mitochondrial energetics, and probably its interaction with PTCD3 participates in this function.
|
14 |
Modelagem molecular, síntese e avaliação da atividade biológica de potenciais antineoplásicos com a proteína hnRNP K e culturas de células tumorais / Molecular modeling, synthesis and biological evaluation of potential antineoplastics with hnRNP K and tumoral cell linesVinicius Barreto da Silva 23 September 2011 (has links)
A proteína hnRNP K é conhecida por seu papel nos múltiplos processos que compõe a expressão gênica, incluindo funções nos estágios de splicing, transcrição e tradução, desempenhadas, principalmente, através da ligação de seus domínios KH a nucleotídeos. A ativação inadequada da hnRNP K tem relação direta com a gênese de alguns tipos de câncer, sobretudo de cabeça e pescoço, mama e colo-retal, evidenciando a mesma como um atrativo alvo molecular para o desenvolvimento de novos fármacos antineoplásicos. Com o auxílio de técnicas in silico, foram identificados dois compostos orgânicos, um derivado de benzimidazol e outro derivado de fenilbenzamida, capazes de impedir a ligação da proteína hnRNP K a oligonucleotídeos in vitro. Aliando as técnicas de docking, campos de interação e dinâmica molecular foi possível sugerir que tais compostos apresentam características estruturais que permitem a realização de interações na fenda de ligação do domínio KH3, principalmente com os resíduos R40 e R59, os quais são considerados chaves no reconhecimento molecular de nucleotídeos. Os derivados de benzimidazol e fenilbenzamida constituem novos compostos de partida na busca de novos antineoplásicos que tem como alvo a proteína hnRNP K. Do ponto de vista de metabolismo e toxicidade, o derivado de fenilbenzamida parece ser mais promissor quando se investiga uma molécula para aplicação terapêutica, uma vez que gerou poucos alertas críticos de toxicidade, ao contrario do derivado de benzimidazol, que apresenta maior potencial genotóxico. Apesar de se ligarem aos domínios KH da hnRNP K e impedir sua complexação a nucelotídeos, ensaios com culturas de células mostraram apenas tênue atividade antitumoral para tais compostos, com maior redução de viabilidade celular, ao redor de 18% a 8,4 M, exibida pelo derivado de fenilbenzamida. Seguindo o princípio do análogo ativo, simulações de triagem virtual na busca de análogos dos ligantes da hnRNP K revelaram 21 novos derivados de benzimidazol ou fenilbenzamida na base de dados EXPRESS-Pick, dos quais 5 foram testados in vitro com a proteína e 3 novos ligantes identificados. Com o intuito de otimizar tais derivados, foram sugeridos in silico substituintes (potenciais bioisósteros) para os anéis dioxopirrolidínicos dos ligantes já identificados, guiando, assim, a futura síntese de novas substâncias com potencial atividade antitumoral. Além disso, o trabalho foi complementado através da proposição de síntese de novos derivados benzoxazepínicos acoplados a purinas, os quais também tem aplicação como antineoplásicos, entretanto por mecanismos que não envolvem a hnRNP K. A grande limitação desses derivados é a presença de um grupo nitro aromático, o qual é reconhecido por sua toxicidade pronunciada. Com o intuito de otimizar tais derivados, foram sugeridos in silico potenciais bioisósteros capazes de substituir o grupo nitro e guiar a síntese e novos derivados com atividade antitumoral e toxicidade reduzida. / hnRNP K protein is known for its role in the multiple processes that compose gene expression, including functions during splicing, transcription and translation, developed, mainly, by the binding of nucleotides to KH domains. Inadequate activation of hnRNP K induces the development of some types of cancer, including head and neck, breast and colorectal. In this way, hnRNP K is an attractive molecular target for antineoplastic drug design. Using in silico strategies, we have identified two organic compounds, a benzimidazole and a phenylbenzamide derivatives, able to prevent the natural binding of nucleotides to hnRNP K in vitro. Applying docking, molecular interaction fields and molecular dynamics simulations it was possible to propose that such compounds present structural characteristics capable to support intermolecular interactions inside KH3 domain binding cleft, mainly with R40 and R59 residues, which are extremely important during molecular recognition of nucleotides by hnRNP K. The benzimidazole and phenylbenzamide derivatives identified are novel lead compounds that can guide the design of new antineoplastic drugs targeting hnRNP K. Considering metabolic and toxicity predictions, the phenylbenzamide seems to be more promising than the benzimidazole derivative as a drug, once the benzimidazole presents genotoxic potential. Although both derivatives prevent the binding of nucleotides to hnRNP K, biological assays with tongue cancer cell lines revealed only a mild antitumoral activity for such compounds. Higher level of cell viability reduction, 18% at 8.4 M, was observed for the phenylbenzamide derivative. Following the similarity principle, virtual screening simulations were made intending to find novel benzimidazole and phenylbenzamide derivatives inside EXPRESS-Pick database. The search revealed 21 compounds, 5 of which were tested in vitro with hnRNP K, where 3 of them were active. Intending to optimize benzimidazole and phenylbenzamide derivatives in order to design more potent chemical entities, we have suggested in silico substituents as potential bioisosteric groups of the dioxopyrrolidine rings of hnRNP K ligands, guiding the future synthesis of novel compounds with enhanced antitumoral activity. Moreover, complementary work proposition was performed through the synthesis of benzoxazepin-purines, which also present antitumoral activity but not through hnRNP K pathway. The major limitation of such derivatives is the presence of a nitro aromatic group, which can be very toxic. 20 potential bioisosteric groups were proposed as fragment candidates to replace the nitro one in order to design novel antitumoral derivatives with reduced toxic potential.
|
15 |
Análise da proteína hnRNP K nas linhagens celulares NB4 e NB4-R2 de leucemia promielocítica aguda com ênfase na patogênese e na diferenciação celular pelo ácido all-trans retinoico / Analysis of hnRNPK protein in cell lines NB4 and NB4-R2 of acute promyelocytic leukemia with emphasis in pathogenesis and cell differentiation by all-trans retinoic acidPadovani, Karina Stringhetta 20 March 2017 (has links)
A ribonucleoproteína heterogênea nuclear K (hnRNP K) e o inibidor endógeno da fosfatase 2A (SET) são superexpressos e propostos como marcadores prognósticos em leucemia mieloide aguda e crônica. O objetivo do estudo foi caracterizar a participação das proteínas hnRNP K e SET na leucemogênese da leucemia promielocítica aguda (LPA), assim como na diferenciação celular induzida pelo ácido all-trans retinóico (ATRA). Os resultados iniciais de qRT-PCR demonstram que os níveis de RNAm de HNRNPK e SET estão aumentados em pacientes ao diagnóstico de LPA em comparação com amostras de indivíduos saudáveis e diminuem após indução e durante a manutenção do tratamento. Os resultados foram validados por Western blot, sugerindo hnRNP K e SET como marcadores diagnóstico e de resposta ao tratamento. O knockdown de hnRNP K e SET foi realizado em células de LPA sensível, NB4, e resistente ao ATRA, NB4-R2, utilizando RNA de interferência. Ambas as proteínas também foram testadas como alvo terapêutico com a utilização de inibidores de hnRNP K (U0126) e SET (OP449 e FTY720). A diminuição de hnRNP K nas células levou ao aumento da diferenciação celular granulocítica em ambas as células, principalmente na presença de ATRA, e portanto, foi capaz de reverter o fenótipo de resistência ao ATRA das células NB4-R2. O knockdown de hnRNP K, assim como o tratamento com U0126, levou a perda de viabilidade dessas células por indução de apoptose acompanhada da clivagem da proteína SET. O knockdown de SET em células LPA confirmou que a indução de apoptose em células com knockdown de hnRNP K ocorreu por clivagem e não pela diminuição da proteína SET nas células. Além disso, demonstrou também que SET prejudica a atuação do ATRA no processo de diferenciação celular. O modelo in vivo utilizando transplante de NB4-R2 em camundongos nude confirmou que o trióxido de arsênico (ATO) combinado a U0126 tem um maior potencial contra a progressão tumoral quando comparado ao tratamento isolado com ATO. FTY720 e OP449 tiveram efeito anti-leucêmico significativo com redução da viabilidade celular. Quando em associação, FTY720 e OP449, apresentaram efeito sinérgico significativo em NB4-R2 (CID<0,7). Concluímos que a superexpressão de hnRNP K e SET contribui para o bloqueio da diferenciação celular em promielócitos e prejudicam a atuação do ATRA no tratamento da LPA e, portanto, hnRNPK em associação com a proteína SET representam alvo terapêutico em potencial para terapia anti-leucêmica da LPA, principalmente para pacientes resistentes ao ATRA / Heterogeneous nuclear ribonucleoprotein K (hnRNP K) and endogenous inhibitor of phosphatase 2A (SET) are overexpressed and proposed as prognostic markers in acute and chronic myeloid leukemia. The study aim was to characterize the hnRNP K and SET proteins involvement in acute promyelocytic leukemia leukemia (APL) leukemogenesis as well as all-trans retinoic acid (ATRA) induced cell differentiation. Initial qRT-PCR results demonstrate that HNRNPK and SET mRNA levels are increased in patients diagnosed with APL compare to samples from healthy donors and decrease after induction and during maintenance of treatment. The results were validated by Western blot, suggesting hnRNP K and SET as diagnostic and response to treatment markers. The knockdown of hnRNP K and SET was performed on sensitive, NB4, and ATRA-resistant, NB4-R2, LPA cells using interfering RNA. Both proteins were also tested as a therapeutic target with a use of hnRNP K (U0126) and SET inhibitors (OP449 and FTY720). The decrease of hnRNP K in cells led to increased granulocyte cell differentiation in both cells, especially in the presence of ATRA, and thus was able to reverse the NB4-R2 cells resistance to ATRA phenotype. The hnRNP K knockdown, as well as the treatment with U0126, had a loss of cell viability by induction of apoptosis accompanied by cleavage of the SET protein. The SET knockdown in APL cells confirmed that an induction of apoptosis in cells with hnRNP K knockdown occurred by cleavage and not by the SET protein decrease in the cells. Furthermore, it has also shown that SET impairs the ATRA\'s performance in the cellular differentiation process. The in vivo model using NB4-R2 transplant in nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has a greater potential against tumor progression compared to the treatment isolated with ATO. FTY720 and OP449 have significant anti-leukemic effect reducing cell viability. When in combination, FTY720 and OP449, they had a significant synergistic effect on NB4-R2 (CDI <0.7). We conclude that overexpression of hnRNP K and SET contributes to block cell differentiation in promyelocytes and impair the performance of ATRA in the treatment of APL and therefore hnRNPK in association with a SET protein represent a potential therapeutic target for anti-leukemic therapy of APL, mainly for patients resistant to ATRA
|
16 |
Acúmulo da ribonucleoproteína heterogênea nuclear K em câncer de cabeça e pescoço: estudos mitocondriais / Accumulation of heterogeneous nuclear ribonucleoprotein K in head and neck cancer: mitochondrial studiesCristiana Bernadelli Garcia 03 April 2014 (has links)
A ribonucleoproteína heterogênea nuclear K (hnRNP K) é uma proteína envolvida em processos de expressão gênica e tem sido proposta como ligante de RNAs mensageiros mitocondriais. Apesar de ser considerada um marcador de pior prognóstico no câncer de cabeça e pescoço, o papel da hnRNP K nesta doença ainda é pouco conhecido. O objetivo deste trabalho foi estudar o envolvimento da hnRNP K na mitocôndria com ênfase na bioenergética e na identificação de novos potenciais ligantes de hnRNP K. As linhagens celulares utilizadas foram de carcinoma de cabeça e pescoço (HN13 e CAL 27) com silenciamento de RNA para hnRNP K e células HEK293 com super-expressão de hnRNP K. O efeito do acúmulo celular da hnRNP K na cadeia transportadora de elétrons mitocondrial foi avaliado por meio da atividade dos complexos mitocondriais I, II e V em células HN13. A redução do nível de hnRNP K usando RNA de interferência promoveu uma diminuição da atividade dos complexos nas células HN13, indicando o envolvimento da proteína na eficiência do transporte de elétrons na cadeia respiratória mitocondrial. Células HEK293 com super-expressão da hnRNP K (HEK293/hnRNP K) e as linhagens HN13 e CAL 27 com silenciamento e redução estável de hnRNP K foram utilizadas para determinar o papel de hnRNP K no potencial de membrana mitocondrial, níveis de ATP, produção de lactato e consumo de oxigênio. Células HEK293/hnRNP K comparadas ao controle apresentaram maior nível de ATP, menor potencial de membrana mitocondrial, menor consumo de oxigênio e maior produção de lactato. As células HN13 com redução da hnRNP K apresentaram níveis mais baixos de ATP, com menor liberação de lactato para o meio extracelular e maior consumo de oxigênio. Esses resultados sugerem que o acúmulo da proteína hnRNP K tem ação importante na mitocôndria por alterar o metabolismo bioenergético celular de fosforilação oxidativa para glicólise anaeróbica. A estratégia de co-imunoprecipitação usando anticorpos para hnRNP K, digestão de proteínas com tripsina e cromatografia líquida acoplada a espectrômetria de massa foi usada para encontrar novos potenciais ligantes de hnRNP K. A análise dos dados com o software SEPro identificou 57 proteínas candidatas a ligantes da hnRNP K. Três proteínas foram validadas por co-IP e Western blotting: o fator de transcrição mitocondrial PTCD3, YB1 e PSF. Propomos que a hnRNP K apresenta função na energética mitocondrial, e provavelmente, a sua interação com PTCD3 participa desta função. / Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a protein involved in gene expression processes, which has been proposed to bind mitochondrial mRNAs. Despite it to be considered a prognostic marker in cancer, the hnRNPK role in this disease is unknown. We addressed the involvement of hnRNP K in mitochondria with emphasis on bioenergetics and identification of new potential ligands of hnRNP K. The cell lines used were from head and neck squamous cell carcinoma (HN13 and CAL 27) with RNA silencing for hnRNP K , and HEK293 cells with overexpression of hnRNP K. The effects of cellular accumulation of hnRNP K in mitochondrial electron chain carriers were assessed by the activity of mitochondrial complexes I, II and V in HN13 cells. Reduced levels of hnRNP K using RNA interference promoted a decrease in the activity of the complexes in HN13 cells, indicating the involvement of the protein in the efficiency of the electron transport in mitochondrial respiratory chain. HEK293 cells with overexpression of hnRNP K (HEK293/hnRNP K) and HN13 and CAL 27 cells with silencing and stable reduction of hnRNP K were used to determine the role of hnRNP K in mitochondrial membrane potential, ATP levels, lactate production and oxygen consumption. HEK293/hnRNP K, compared to control cells, showed higher levels of ATP, reduced mitochondrial membrane potential, lower oxygen consumption and higher production of lactate. HN13 cells with reduced hnRNP K had lower ATP levels, with lower release of lactate to the extracellular medium and higher oxygen consumption. These results suggest that accumulation of hnRNP K protein plays a role in mitochondria by changing the cellular energetic metabolism from oxidative phosphorylation to glycolysis. The strategy of co-immunoprecipitation using antibodies for hnRNP K, protein digestion with trypsin, and liquid chromatography, coupled to mass spectrometer, were used to search for new potential ligands of hnRNP K. Data analysis with software SEPro identified 57 candidate proteins binding to hnRNP K. Three proteins were validated by co-IP and Western blotting: the mitochondrial transcription factor PTCD3, YB1, and PSF. We propose that hnRNP K plays a role in the mitochondrial energetics, and probably its interaction with PTCD3 participates in this function.
|
17 |
Hypertension et régulation de l'expression moléculaire de l'angiotensinogène par la ribonucléoprotéine hétérogène nucléaire KAbdo, Shaaban 06 1900 (has links)
Le diabète est une maladie chronique dont la principale
caractéristique est un niveau plasmatique élevé de glucose, qui est causé
soit par un défaut dans la production d’insuline, l’action de l’insuline, ou
les deux à la fois. Plusieurs études ont démontré que l’hyperglycémie
chronique peut mener à la dysfonction et même la défaillance de
plusieurs organes, dont le coeur, le système vasculaire, les yeux et les
reins, se traduisant par des infarctus du myocarde, des accidents
cérébro-vasculaires et des complications rétinales et rénales,
respectivement. La néphropathie diabétique (DN) est la principale cause
de déficience rénale et affecte près de 25-40% des patients diabétiques.
La DN est invariablement associée à un risque élevé d’accident cérébrovasculaire
et de dysfonction cardivasculaire. L’angiotensinogène (Agt) est
l’unique précurseur de tous les types d’angiotensines. En plus du
système rénine-angiotensine (RAS) sytémique, le rein possède son
propre système intrarénal et exprime tous les composants du RAS. L’Agt
est fortement exprimé dans les cellules du tubule proximal rénal (RPTC)
et y est converti en angiotensine II (AngII), le peptide biologiquement actif
du RAS. Les patients diabétiques présentent de hauts niveaux d’AngII et
une augmentation de l’expression des gènes du RAS, suggérant que
l’activation du RAS intrarénal joue un rôle important dans la progression
de la DN. Les mécanismes qui contrôlent la régulation du niveau rénal
d’Agt par l’hyperglycémie et l’insuline demeurent mal compris.
Le but global de cette thèse est de mieux comprendre les
mécanismes moléculaires qui contrôlent l’expression du gène Agt chez la
souris Akita (un modèle murin de diabète de type 1). Dans cette optique,
la première partie de la thèse se concentre sur deux facteurs de
transcription de la famille des ribonucléoprotéines nucléaires hétérogènes
(hnRNP). Chan et collaborateurs ont déjà identifié 2 protéines nucléaires
hnRNP F et hnRNP K, de 48kD et 70kD respectivement. HnRNP F et
hnRNP K forment un hétérodimère et se lient à l’élément de réponse à
l’insuline (IRE) présent dans le promoteur du gène Agt du rat et inhibent
la transcription du gène Agt in vitro. Afin de déterminer si hnRNP F / K
sont responsables de l’inhibition de l’expression rénale de Agt par
l’insuline in vivo, nous avons étudié des souris Akita males traités ou non
avec des implants d’insuline pour une période de 4 semaines. Des souris
non-Akita males ont été employées comme contrôles. Les souris Akita
développent de l’hypertension et de l’hypertrophie rénale. Le traitement à
l’insuline rétablit les niveaux de glucose plasmatiques et la pression
systolique (SBP), et atténue l’hypertrophie rénale, l’albuminurie (ratio
albumine/créatinine urinaire, ACR) et les niveaux urinaires d’Agt et AngII
chez les souris Akita. De plus, le traitement à l’insuline inhibe l’expression
rénale du gène Agt, tout en augmentant l’expression des gènes hnRNP
F, hnRNP K et ACE2 (enzyme de conversion de l’angiotensine-2). Dans
des RPTC in vitro, l’insuline inhibe Agt, mais stimule l’expression de
hnRNP F et hnRNP K en présence de hautes concentrations de glucose,
et ce via la voie de signalisation MAPK p44/42 (protéine kinase activée
par un mitogène). La transfection avec des petits ARN interférents
(siRNA) contre hnRNP F et hnRNP K prévient l’inhibition de l’expression
d’Agt par l’insuline dans les RPTC. Cette étude démontre bien que
l’insuline prévient l’hypertension et atténue les dommages rénaux
observés chez les souris Akita diabétiques, en partie grâce à la
suppression de la transcription rénale de Agt, via une augmentation de
l’expression de hnRNP F et hnRNP K.
La seconde partie de cette thèse change de focus et se tourne
vers le facteur Nrf2 (nuclear factor erythroid 2-related factor 2). Nrf2 est
un facteur de transcription qui contrôle les gènes de la réponse
antioxydante cellulaire en réponse au stress oxydant ou aux
électrophiles. Le but de cette étude est d’examiner l’impact de la
surexpression de la catalase (Cat) dans les RPTC sur l’expression du
gène Agt via Nrf2 et sur le développement de l’hypertension et des
dommages rénaux résultants chez les souris diabétiques Akita
transgéniques (Tg). Nos études ont démontré que la surexpression de
Cat dans les souris Akita Cat-Tg normalise la SBP, atténue les
dommages rénaux et inhibe l’expression des gènes Nrf2 et Agt dans les
RPTC. In vitro, le glucose élevé (HG) et l’oltipraz (un activateur de Nrf2)
stimulent l’expression de Nrf2 et Agt, et cet effet peut être bloqué par la
trigonelline (inhibiteur de Nrf2), des siRNA contre Nrf2, des antioxydants
ou des inhibiteurs pharmacologiques NF-κB et MAPK p38. La
suppression de sites de réponse à Nrf2 présents dans le promoteur du
gène Agt du rat abolit la stimulation par l’oltipraz. Finalement, des souris
males adultes non-transgéniques traitées avec l’oltipraz montrent une
augmentation de l’expression de Nrf2 et Agt dans leurs RPTC et cette
augmentation peut être normalisée par la trigonelline. Ces données
permettent d’identifier un nouveau mécanisme d’action de Nrf2, par la
stimulation du gène Agt intrarénal et l’activation du RAS, qui induisent
l’hypertension et les dommages rénaux par le glucose élevé et les
espèces réactives de l’oxygène chez les souris diabétiques. Nos
conclusions permettent de démontrer que l’insuline induit l’expression de
hnRNP F et hnRNP K, qui jouent ensuite un rôle protecteur en prévenant
l’hypertension. La surexpression de la catalase dans les RPTC vient
quant à elle atténuer l’activation de Nrf2 et ainsi réduit la SBP chez les
souris Akita. / Diabetes mellitus is a chronic metabolic disorder characterized by
high plasma glucose caused by an impairment of insulin production,
insulin action or both. Accumulating evidence has shown that chronic
hyperglycemia can lead to dysfunction and failure of multiple organs
including the heart, vascular system, eyes, and kidneys resulting in
myocardial infarction, stroke, and retinal and renal complications,
respectively. Diabetic nephropathy (DN) is the leading cause of end-stage
renal disease affecting approximately 25–40% of diabetic patients. DN is
invariably associated with an increased risk of stroke and cardiovascular
dysfunction. Angiotensinogen (Agt) is the sole precursor for all types of
angiotensins. In addition to systemic renin-angiotensin system (RAS), all
the components of the intrarenal RAS are expressed in the kidney. Agt is
highly expressed in the renal proximal tubular cells (RPTCs) and
converted into biologically active angiotensin II (Ang II). In Diabetics,
intrarenal Ang II level and RAS gene expression are upregulated,
suggesting that intrarenal RAS activation plays an important role in the
progression of DN. The mechanism (s) underlying the regulation of renal
Agt by hyperglycemia and insulin are not completely understood. The
overall aim of this thesis is to understand the molecular mechanism(s)
that regulate renal Agt gene expression in an Akita mouse (a mouse
model of type 1 diabetes). For this purpose, the first part of this thesis
focuses on two transcription factors from the heterogenous nuclear
ribonucleoprotein (hnRNPs) family. Previously, Chan’s group identified
two nuclear proteins hnRNP F and hnRNP K of 48kD and 70kD,
respectively. hnRNP F and hnRNP K form a heterodimer and bind to the
insulin-responsive element (IRE) in the rat Agt gene promoter inhibiting
Agt gene transcription in vitro. To determine whether hnRNP F / K
mediate insulin inhibition of renal Agt expression in vivo, we used adult
male Akita mice treated ± insulin implants for 4 weeks. Non-Akita mice
served as controls. The Akita mice developed hypertension and exhibited
renal hypertrophy. Insulin treatment normalized plasma glucose levels
and systolic blood pressure (SBP), attenuated renal hypertrophy,
decreased urinary albumin/creatinine ratio (ACR) and urinary Agt and
Ang II levels in Akita mice. Furthermore, insulin treatment inhibited renal
Agt expression but enhanced hnRNP F, hnRNP K and angiotensinconverting
enzyme-2 (ACE2) expression. In vitro, insulin inhibited Agt but
stimulated hnRNP F and hnRNP K expression in high-glucose media via
p44/42 mitogen-activated protein kinase signaling in RPTCs. Transfection
with hnRNP F and hnRNP K small interfering RNAs (siRNA) prevented
the insulin inhibition of Agt expression in RPTCs. This study
demonstrates that insulin prevents hypertension and attenuates kidney
injury, at least in part, through suppressing renal Agt transcription via
upregulation of hnRNP F and hnRNP K expression in diabetic Akita mice.
In the second part of the thesis we focused on the nuclear factor
erythroid 2-related factor 2 (Nrf2). Nrf2 is a transcription factor that
regulates cellular antioxidant gene defense against oxidative stress or
electrophiles. The purpose of this study is to investigate the impact of the
overexpressing catalase (Cat) in RPTCs on Agt gene expression via
Nrf2and the resulting effects on the development of hypertension and
renal injury in diabetic Akita transgenic (Tg) mice. Our studies
demonstrate that Cat overexpression normalizes SBP, attenuates renal
injury, and inhibits RPTC Nrf2 and Agt gene expression in the Akita Cat-
Tg compared to Akita mice. In vitro, high glucose (HG) and Oltipraz
stimulated Nrf2 and Agt gene expression; these changes were blocked by
Trigonelline (an inhibitor of Nrf2), siRNA against Nrf2, antioxidants, or
pharmacological inhibitors of NF-kB and p38 mitogen-activated protein
kinase. Moreover, deletion of Nrf2-responsive elements in the rat Agt
gene promoter abolishes the stimulatory effect of Oltipraz. Finally,non
transgenic adult male mice treated with the Nrf2 activator Oltipraz,
upregulated Nrf2 and Agt expression in mouse RPTs, an effect that was
normalized by Trigonelline. These data identify a novel mechanism via
which Nrf2 mediates the stimulation of intrarenal Agt gene expression and
activates the RAS through whichHG/reactive oxygen species (ROS)
induce hypertension and renal injury in diabetic mice. Our findings
demonstrate that the insulin induced hnRNP F and hnRNP K gene
expression play a protective role in the preventing hypertension. Catalase
overexpression, in RPT's, attenuates Nrf2 activation and lowers the SBP
in Akita mice.
|
18 |
The Regulation of the Alternative Splicing of Caspase 9Goehe, Rachel 24 September 2010 (has links)
The pro-apoptotic, caspase 9a, and the anti-apoptotic, caspase 9b, are derived from the caspase 9 gene by alternative splicing. This study demonstrates that the alternative splicing of caspase 9 is dysregulated in a large percentage of non-small cell lung cancer (NSCLC) tumors of the adenocarcinoma type. Furthermore, modulation of the levels of splice variants of caspase 9 had dramatic effects on the anchorage-independent growth and tumorigenic capacity of NSCLC cells. Due to these findings, the molecular mechanisms regulating the post-transcriptional processing of caspase 9 were therefore examined and an exonic splicing silencer (ESS) regulating the pre-mRNA processing of caspase 9 was identified. To study the possible RNA trans-factors interacting with this RNA sequence, we utilized an electromobility shift assay (EMSA) coupled with competitor studies and demonstrated three specific protein:RNA complexes for this ESS. Affinity purification and mass spectrometry analysis identified hnRNP L as part of these protein:RNA complexes. Downregulation of hnRNP L induced a significant increase in caspase 9a/caspase 9b mRNA ratio, which translated to the protein level. Expression of hnRNP L verified the siRNA specificity lowering the caspase 9a/9b ratio, but expression of hnRNP L produced the contrasting effect in non-transformed cells suggesting a post-translational modification specific for NSCLC cells. Indeed, the phospho-status of hnRNP L was significantly increased in NSCLC cells, and mutagenesis studies identified Ser52 as a critical residue regulating the ability of hnRNP L to repress the inclusion of the exon 3,4,5,6 cassette into the mature caspase 9 mRNA. The biological relevance of this mechanism was demonstrated by stable downregulation of hnRNP L in NSCLC cells, which induced a complete loss of both anchorage-independent growth and tumorigenic capacity. This effect of hnRNP L downregulation was due to distal modulation of the alternative splicing of caspase 9 as the loss of both phenotypes was “rescued” by ectopic expression of caspase 9b. Therefore, this study identifies cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase 9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.
|
19 |
Regulation of alternative pre-mRNA splicing by depolarization/CaMKIVLiu, Guodong 29 June 2012 (has links)
Alternative pre-mRNA splicing is often controlled by cell signals (1-3). Membrane depolarization/calcium (Ca2+) signaling controls alternative splicing of a group of genes in neurons and endocrine cells (4-9), with important implications in memory formation or secretion of hormones and neurotransmitters (10-15). However, the underlying molecular basis remains largely unknown.
In rat GH3 pituitary cells, BK potassium channels control cellular electrical firing, which is critical for the release of growth hormone and prolactin. Inclusion of the STREX exon of the Slo1 gene encoding the channel α subunit is repressed by the Ca2+/calmodulin-dependent kinase IV (CaMKIV) upon depolarization (4). We isolated CaMKIV-responsive RNA elements (CaRREs) from a library of 13-nucleotide random sequences through in vivo selection in HEK293T cells. Most elements are CA-rich or A-rich, with the heterogeneous nuclear ribonucleoprotein (hnRNP) L as a binding factor. This is consistent with the finding that CA-rich elements and hnRNP L are targeted by CaMKIV in the regulation of splicing (16).
In further efforts to directly link the kinase with hnRNP L, we showed that hnRNP L is essential for the full repression of STREX by depolarization and that a highly conserved CaMKIV target serine (Ser513) of L is required. Ser513 phosphorylation enhanced L binding to the STREX CaRRE1, leading to reduced binding of the constitutive factor U2AF65 to the 3’ splice site of STREX. Mutation of Ser513 abolished both activities. Therefore, hnRNP L mediates the repression of STREX by depolarization through modulation of a key step in spliceosomal assembly.
We further identified hnRNP L, L-like (LL) and PTB as repressors of STREX and other depolarization-regulated exons with differential effects. Moreover, a full response of STREX to depolarization is mediated by combinations of hnRNP L and LL or PTB. Another depolarization-responsive exon, the exon 18 of the neuregulin 1 gene, is also controlled in a similar way, with the hnRNP L Ser513 required as well.
This work provides the first direct link between the Ca2+ signaling and a specific serine of a regulatory splicing factor. Elucidation of the underlying molecular mechanisms would likely help us understand the fine-tuning of hormone secretion and memory formation.
|
20 |
Regulation of alternative pre-mRNA splicing by depolarization/CaMKIVLiu, Guodong 29 June 2012 (has links)
Alternative pre-mRNA splicing is often controlled by cell signals (1-3). Membrane depolarization/calcium (Ca2+) signaling controls alternative splicing of a group of genes in neurons and endocrine cells (4-9), with important implications in memory formation or secretion of hormones and neurotransmitters (10-15). However, the underlying molecular basis remains largely unknown.
In rat GH3 pituitary cells, BK potassium channels control cellular electrical firing, which is critical for the release of growth hormone and prolactin. Inclusion of the STREX exon of the Slo1 gene encoding the channel α subunit is repressed by the Ca2+/calmodulin-dependent kinase IV (CaMKIV) upon depolarization (4). We isolated CaMKIV-responsive RNA elements (CaRREs) from a library of 13-nucleotide random sequences through in vivo selection in HEK293T cells. Most elements are CA-rich or A-rich, with the heterogeneous nuclear ribonucleoprotein (hnRNP) L as a binding factor. This is consistent with the finding that CA-rich elements and hnRNP L are targeted by CaMKIV in the regulation of splicing (16).
In further efforts to directly link the kinase with hnRNP L, we showed that hnRNP L is essential for the full repression of STREX by depolarization and that a highly conserved CaMKIV target serine (Ser513) of L is required. Ser513 phosphorylation enhanced L binding to the STREX CaRRE1, leading to reduced binding of the constitutive factor U2AF65 to the 3’ splice site of STREX. Mutation of Ser513 abolished both activities. Therefore, hnRNP L mediates the repression of STREX by depolarization through modulation of a key step in spliceosomal assembly.
We further identified hnRNP L, L-like (LL) and PTB as repressors of STREX and other depolarization-regulated exons with differential effects. Moreover, a full response of STREX to depolarization is mediated by combinations of hnRNP L and LL or PTB. Another depolarization-responsive exon, the exon 18 of the neuregulin 1 gene, is also controlled in a similar way, with the hnRNP L Ser513 required as well.
This work provides the first direct link between the Ca2+ signaling and a specific serine of a regulatory splicing factor. Elucidation of the underlying molecular mechanisms would likely help us understand the fine-tuning of hormone secretion and memory formation.
|
Page generated in 0.4684 seconds