RESUMO
FK866 is a specific inhibitor of NAMPT and induces apoptosis of leukemic cells by depletion of intracellular NAD(+). Since up-regulation of NAMPT is associated with several cases of cancers, including leukemias, we asked whether in leukemic cells inhibition of NAMPT involves p53 pathway. We observed that FK866 induced apoptosis and reduced cell proliferation in NB-4, OCI-AML3 and MOLM-13 cell lines. In contrast, the leukemia cell lines, K-562 and Kasumi, containing nonfunctional p53 were relatively unaffected by FK866 treatment. Importantly, direct inhibition of sirtuins significantly reduced the viability of NB-4, OCI-AML3 and MOLM-13 cell lines. Activation of p53 by FK866 involved increased acetylation of p53 at lysine 382 with subsequent increase in the expression of p21 and BAX. Further, knockdown of p53 attenuated the effects of FK866 on apoptosis and cell cycle arrest, which was partly associated with decreased expression of p21 and BAX. Our results suggest the role of p53 acetylation pathway in the anti-leukemic effect of FK866.
Assuntos
Acrilamidas/farmacologia , Apoptose/efeitos dos fármacos , Citocinas/antagonistas & inibidores , NAD/deficiência , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Técnicas de Inativação de Genes , Humanos , Leucemia Mieloide , Sirtuínas/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossínteseRESUMO
Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.
Assuntos
Acrilamidas/farmacologia , Apoptose/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Proteína Supressora de Tumor p53/agonistas , Acetilação , Ciclo Celular , Citocinas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lisina/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Neoplasias/enzimologia , Neoplasias/virologia , Nicotinamida Fosforribosiltransferase/metabolismo , Vírus 40 dos Símios , Sirtuína 1/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human immunodeficiency virus-1 (HIV-1) disease is characterized by a relentless decline in CD4(+) T cells, resulting in the development of AIDS. Extracellular Tat secreted from the HIV-1 infected cells, enters non-infected T cells to induce apoptosis. A number of mechanisms, none of which is mutually exclusive, have been attributed to the cell depletion property of Tat protein. In the present communication, we provide evidence that the cell-killing effect of Tat is mediated by the activation of p53 pathway via inhibition of SIRT1, an NAD(+)-dependent deacetylase belonging to class III histone deacetylases. This evidence is based on the following experimental facts reported herein: (1) Overexpression of Tat protein decreases both the deacetylase and promoter activity of SIRT1, (2) SIRT1 inhibition by Tat involves increased levels of acetylated p53 and (3) The activation of p53 leads to subsequent increases in the expression of p53 target genes, p21 and BAX.
Assuntos
HIV-1/metabolismo , Sirtuína 1/antagonistas & inibidores , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Acetilação , Antígenos CD4/análise , Inibidor de Quinase Dependente de Ciclina p21/genética , Células HEK293 , Células HeLa , Humanos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Sirtuína 1/genética , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Nicotinamide-phosphoribosyltransferase (NAMPT), induced under stress, converts nicotinamide (NA) to nicotinamide mononucleotide (NMN), which then reacts with ATP to regenerate NAD(+). Despite the pivotal role of NAD(+) in metabolic reactions, the molecular pathways triggered by the intracellular changes in NAD(+) level in cancer cells are largely unknown. Growth Arrest and DNA Damage-inducible Gene (GADD45A) is regulated by multiple cellular factors which play an important role in the control of cell-cycle checkpoint, DNA repair process and signal transduction. The present study was designed to assess the significance of intracellular NAD(+) levels on the regulation of GADD45A expression. The results of this study demonstrate an inverse relationship between NAMPT expression and the regulation of GADD45A gene. Thus, an overexpression of NAMPT led to a decreased expression of GADD45A, whereas, the inhibition of NAMPT by the known chemical inhibitor FK866 increased the expression of GADD45A in cells. Inhibition of SIRT1, an NAD(+)-dependent deacetylase, using shRNA also led to an increased expression of GADD45A gene. In further experiments we could show that the increased expression of GADD45A under the above experimental conditions, NAMPT inhibition by FK866, involves acetylation of FOXO3a, a member of the important family of forkhead (FOXO) proteins. This knowledge should contribute to our understanding of the role played by NAMPT and SIRT1 in the regulation of GADD45A expression by FOXO3a.