RESUMO
CML is effectively treated with tyrosine kinase inhibitors (TKIs). However, the efficacy of these drugs is confined to the chronic phase of the disease and development of resistance to TKIs remains a pressing issue. The anti-inflammatory COX2 inhibitor celecoxib has been utilized as anti-tumour drug due to its anti-proliferative activity. However, its effects in hematological malignancies, in particular CML, have not been investigated yet. Thus, we tested biological effects and mechanisms of action of celecoxib in Philadelphia-positive (Ph+) CML and ALL cells.We show here that celecoxib suppresses the growth of Ph+ cell lines by increasing G1-phase and apoptotic cells and reducing S- and G2-phase cells. These effects were independent of COX2 inhibition but required the rapid activation of AMP-activated protein kinase (AMPK) and the consequent inhibition mTORC1 and 2. Treatment with celecoxib also restored GSK3ß function and led to down-regulation of ß-catenin activity through transcriptional and post-translational mechanisms, two effects likely to contribute to Ph+ cell growth suppression by celecoxib.Celecoxib inhibited colony formation of TKI-resistant Ph+ cell lines including those with the T315I BCR-ABL mutation and acted synergistically with imatinib in suppressing colony formation of TKI-sensitive Ph+ cell lines. Finally, it suppressed colony formation of CD34+ cells from CML patients, while sparing most CD34+ progenitors from healthy donors, and induced apoptosis of primary Ph+ ALL cells.Together, these findings indicate that celecoxib may serve as a COX2-independent lead compound to simultaneously target the mTOR and ß-catenin pathways, key players in the resistance of CML stem cells to TKIs.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Celecoxib/farmacologia , Proliferação de Células/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , beta Catenina/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Proteínas de Fusão bcr-abl/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HeLa , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Células Tumorais Cultivadas , beta Catenina/genéticaRESUMO
High plasma levels of nicotinamide phosphoribosyltransferase (NAMPT), traditionally considered an intracellular enzyme with a key role in NAD synthesis, have been reported in several oncological, inflammatory and metabolic diseases. We now show that eNAMPT can be actively released by melanoma cells in vitro. We analysed the mechanisms of its release, and we found both classical and non-classical pathway involvement. eNAMPT released by melanoma cells, in our hands, has paracrine and autocrine effects: it activates MAPK, AKT and NF-κB pathways and increases colony formation in anchorage-independent conditions. eNAMPT also induces M1 polarization in human monocytes. Last, we demonstrate, for the first time in any cancer type, that eNAMPT levels in plasma of tumour-bearing mice increase and that this increase can be reconducted to the tumour itself. This provides an important cue on previous observations that eNAMPT is increased in patients with cancer. Moreover, silencing NAMPT in melanoma cells leads to a reduction in the tumour growth rate. Our findings extend the basis to consider eNAMPT as a cytokine involved in tumour progression.
Assuntos
Citocinas/metabolismo , Melanoma/enzimologia , Nicotinamida Fosforribosiltransferase/metabolismo , Neoplasias Cutâneas/enzimologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/sangue , Espaço Extracelular/enzimologia , Humanos , Peróxido de Hidrogênio/farmacologia , Melanoma/patologia , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Nicotinamida Fosforribosiltransferase/sangue , Comunicação Parácrina/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
The transcription factor MycN is the prototypical neuroblastoma oncogene and a potential therapeutic target. However, its strong expression caused by gene amplification in about 30% of neuroblastoma patients is a considerable obstacle to the development of therapeutic approaches aiming at eliminating its tumourigenic activity. We have previously reported that B-Myb is essentially required for transcription of the MYCN amplicon and have also shown that B-MYB and MYCN are engaged in a feed forward loop promoting the survival/proliferation of neuroblastoma cells. We postulated that pharmacological strategies breaking the B-MYB/MYCN axis should result in clinically desirable effects. Thus, we implemented a high throughput chemical screen, using a curated library of ~1500 compounds from the National Cancer Institute, whose endpoint was the identification of small molecules that inhibited B-Myb. At the end of the screening, we found that the compounds pinafide, ellipticine and camptothecin inhibited B-Myb transcriptional activity in luciferase assays. One of the compounds, the topoisomerase-1 inhibitor camptothecin, is of considerable clinical interest since its derivatives topotecan and irinotecan are currently used as first and second line treatment agents for various types of cancer, including neuroblastoma. We found that neuroblastoma cells with amplification of MYCN are more sensitive than MYCN negative cells to camptothecin and topotecan killing. Campothecin and topotecan caused selective down-regulation of B-Myb and MycN expression in neuroblastoma cells. Notably, forced overexpression of B-Myb could antagonize the killing effect of topotecan and camptothecin, demonstrating that the transcription factor is a key target of the drugs. These results suggest that camptothecin and its analogues should be more effective in patients whose tumours feature amplification of MYCN and/or overexpression of B-MYB.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Neuroblastoma/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Transativadores/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Camptotecina/análogos & derivados , Camptotecina/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/patologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Bibliotecas de Moléculas Pequenas , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/uso terapêutico , Topotecan/química , Topotecan/uso terapêutico , Transativadores/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Transgenes/genéticaRESUMO
The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell-cell and cell-matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described.