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1.
Front Mol Biosci ; 9: 823195, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35720128

RESUMO

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.

3.
J Biol Chem ; 286(48): 41600-41615, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21862591

RESUMO

Unique sensitivity of tumor cells to the inhibition of glycolysis is a good target for anticancer therapy. Here, we demonstrate that the pharmacologically activated tumor suppressor p53 mediates the inhibition of glycolytic enzymes in cancer cells in vitro and in vivo. We showed that p53 binds to the promoters of metabolic genes and represses their expression, including glucose transporters SLC2A12 (GLUT12) and SLC2A1 (GLUT1). Furthermore, p53-mediated repression of transcription factors c-Myc and HIF1α, key drivers of ATP-generating pathways in tumors, contributed to ATP production block. Inhibition of c-Myc by p53 mediated the ablation of several glycolytic genes in normoxia, whereas in hypoxia down-regulation of HIF1α contributed to this effect. We identified Sp1 as a transcription cofactor cooperating with p53 in the ablation of metabolic genes. Using different approaches, we demonstrated that glycolysis block contributes to the robust induction of apoptosis by p53 in cancer cells. Taken together, our data suggest that tumor-specific reinstatement of p53 function targets the "Achilles heel" of cancer cells (i.e. their dependence on glycolysis), which could contribute to the tumor-selective killing of cancer cells by pharmacologically activated p53.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Glicólise , Neoplasias/enzimologia , Elementos de Resposta , Proteína Supressora de Tumor p53/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/biossíntese , Proteínas Facilitadoras de Transporte de Glucose/genética , Transportador de Glucose Tipo 1/biossíntese , Transportador de Glucose Tipo 1/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/genética , Neoplasias/terapia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/genética
4.
Neurobiol Learn Mem ; 94(2): 158-66, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20452447

RESUMO

The MAPK/ERK pathway plays an important role in the regulation of gene expression during memory formation both in vertebrates and invertebrates. In the mollusk Helix lucorum, serotonin induces activation of MAPK/ERK in the central nervous system (CNS) upon food aversion learning. Such learning depends on a neuronal network in which specialized neurons play distinct roles so that they may exhibit different activation levels of the MAPK/ERK pathway. Here we performed a comparative analysis of MAPK/ERK activation in single neurons of the food-aversion network, focusing both on command neurons, which mediate withdrawal behavior and process information pertaining to the unconditioned stimulus, and on neurons of the procerebrum, the mollusk's olfactory center, which process information from the conditioned stimulus. By means of Western blots designed to detect micro amounts of proteins, we determined MAPK/ERK activation in these neurons and found that after food aversion learning phospho-ERK levels increased significantly in RPa(2/3) command neurons of the right parietal ganglia and in the procerebrum. Such an increase was prevented by injection of PD98095, an inhibitor of the ERK upstream kinase (MEK-1). In contrast, no activation of MAPK/ERK was detected in similar conditions in the corresponding neurons of the left parietal ganglia LPa(2/3). This asymmetry was verified after serotonin application to the CNS in order to mimic learning. Our results thus show that learning involves synchronous and asymmetric serotonin-dependent MAPK/ERK activation. Such an asymmetry may reflect lateralization of memory processes in the mollusk brain.


Assuntos
Aprendizagem da Esquiva/fisiologia , Lateralidade Funcional/fisiologia , Caracois Helix/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neurônios/enzimologia , Análise de Variância , Animais , Sequência de Bases , Condicionamento Clássico/fisiologia , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/enzimologia , Dados de Sequência Molecular , Rede Nervosa/citologia , Rede Nervosa/enzimologia , Sistemas do Segundo Mensageiro/fisiologia , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/fisiologia , Estatísticas não Paramétricas
5.
Cell Cycle ; 9(9): 1847-55, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20436301

RESUMO

Expression of mutant p53 correlates with poor prognosis in many tumors, therefore strategies aimed at reactivation of mutant p53 are likely to provide important benefits for treatment of tumors that are resistant to chemotherapy and radiotherapy. We have previously identified and characterized a small molecule RITA which binds p53 and induces a conformational change which prevents the binding of p53 to several inhibitors, including its own destructor MDM2. In this way, RITA rescues the tumor suppression function of wild type p53. Here, we demonstrate that RITA suppressed the growth and induced apoptosis in human tumor cell lines of a diverse origin carrying mutant p53 proteins. RITA restored transcriptional transactivation and transrepression function of several hot spot p53 mutants. The ability of RITA to rescue the activity of different p53 mutants suggests its generic mechanism of action. Thus, RITA is a promising lead for the development of anti-cancer drugs that reactivate the tumor suppressor function of p53 in cancer cells irrespective whether they express mutant or wild type p53.


Assuntos
Apoptose , Furanos/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/genética
6.
Cancer Cell ; 15(5): 441-53, 2009 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-19411072

RESUMO

Targeting "oncogene addiction" is a promising strategy for anticancer therapy. We report a potent inhibition of crucial oncogenes by p53 upon reactivation by small-molecule RITA in vitro and in vivo. RITA-activated p53 unleashes the transcriptional repression of antiapoptotic proteins Mcl-1, Bcl-2, MAP4, and survivin; blocks the Akt pathway on several levels; and downregulates c-Myc, cyclin E, and beta-catenin. p53 ablates c-Myc expression via several mechanisms at the transcriptional and posttranscriptional level. We show that the threshold for p53-mediated transrepression of survival genes is higher than for transactivation of proapoptotic targets. Inhibition of oncogenes by p53 reduces the cell's ability to buffer proapoptotic signals and elicits robust apoptosis. Our study highlights the role of transcriptional repression for p53-mediated tumor suppression.


Assuntos
Apoptose , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Furanos/farmacologia , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas , beta Catenina/metabolismo
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