RESUMO
Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Inibidores da Topoisomerase/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antraquinonas/uso terapêutico , Antígenos de Neoplasias/metabolismo , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/genética , Leucemia Linfoide/patologia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Camundongos , Inibidores da Topoisomerase/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Estudos de Coortes , Dano ao DNA , Análise Mutacional de DNA , Doxorrubicina/farmacologia , Citometria de Fluxo , Deleção de Genes , Genoma Humano , Histonas/metabolismo , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Prognóstico , Fatores de Processamento de RNA , Receptor Notch1/genética , Proteína Supressora de Tumor p53/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1, a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in paediatric ALL. We showed a potent in vitro cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic features but dependent on high MYC expression and coupled with transcriptional downregulation of multiple pro-survival pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone. Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.
RESUMO
Neuronal cell death and its regulation have been extensively studied as an essential process of both neurodevelopment and neurodegenerative conditions. However it is not clear how circulating hormones influence such processes. Therefore we aimed to determine whether the anti-obesity hormone leptin could promote the survival of murine central and peripheral neurons in vitro. Thus we established primary neuronal cultures of dopaminergic midbrain neurons and trigeminal sensory neurons and induced cell death via either toxic insult or growth factor withdrawal. We demonstrate that leptin promotes the survival of developing peripheral and central neurons via activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3-kinase)/Akt/nuclear factor kappa B (NF-kappaB) -dependent signaling cascades. Specifically, leptin protects dopaminergic midbrain neurons from the apoptotic stimuli, tumor necrosis factor alpha (TNF-alpha) and 6-hydroxydopamine (6-OHDA). In addition, it promotes the survival of postnatal, but not embryonic, trigeminal sensory neurons following neurotrophin withdrawal. Our data reveal a novel neuroprotective role for leptin in the peripheral nervous system while expanding on the known anti-apoptotic role of leptin in the CNS. These findings have important implications for our understanding of neuronal viability.
Assuntos
Encéfalo/efeitos dos fármacos , Leptina/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nervos Periféricos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Nervos Periféricos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externalised PS of apoptotic cells. Here we demonstrate that CD14 does not function preferentially as a PS receptor in apoptotic-cell clearance. Compared with phosphatidylcholine and phosphatidylethanolamine, PS was the least active phospholipid binding to human monocyte-derived macrophages and showed no specificity for soluble or membrane-anchored CD14. Significantly, PS-containing liposomes failed to inhibit CD14-dependent uptake of apoptotic cells by macrophages. PS exposure was, however, found to be insufficient for either CD14-dependent or CD14-independent apoptotic-cell uptake by phagocytes. The additional features that enable apoptotic-cell clearance are derived from mechanisms that can be divorced temporally from those responsible for the morphological features of apoptosis.
Assuntos
Apoptose , Receptores de Lipopolissacarídeos/fisiologia , Macrófagos/metabolismo , Fosfatidilserinas/metabolismo , Anexina A5/farmacologia , Linhagem Celular , Células Cultivadas , Cicloeximida/farmacologia , Eritrócitos/metabolismo , Citometria de Fluxo , Humanos , Leucócitos/metabolismo , Ligantes , Metabolismo dos Lipídeos , Receptores de Lipopolissacarídeos/metabolismo , Lipossomos/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Fagocitose , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Raios UltravioletaRESUMO
The detection of 3-nitro-L-tyrosine residues associated with many disease states, including gastric cancer, has implicated a role for peroxynitrite in vivo, and thus endogenously produced nitric oxide and superoxide. Additionally, dietary nitrate has been suggested to be involved in the pathogenesis of gastric cancer through a mechanism involving reduction to nitrite and subsequent formation of potentially mutagenic nitroso-compounds. Studies have now demonstrated that a multitude of reactive nitrogen species other than peroxynitrite are capable of producing nitrotyrosine. Thus, we have reviewed the evidence that dietary nitrate, amongst other reactive nitrogen species, may contribute to the body burden of nitrotyrosine.
Assuntos
Dieta/efeitos adversos , Nitratos/metabolismo , Nitritos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Animais , Humanos , Óxido Nítrico/metabolismo , Espécies Reativas de Nitrogênio/metabolismoRESUMO
The action of peroxynitrite in vivo has been proposed to account for the involvement of nitrotyrosine in the pathogenesis of many diseases. However, it has been demonstrated that nitrite under acidic conditions, similar to those in the human stomach, also has the ability to nitrate tyrosine. Dietary nitrate is also implicated in the progression of gastritis and gastric cancer and elevated levels of nitrate are found in many disease states in which nitrotyrosine may play a role. Thus, we investigated whether the dietary nitrate intake might contribute towards the plasma protein-bound levels of nitrotyrosine. Seven healthy, non-smokers participated in a two-day study consisting of a nitrate-low control day followed by a day during which three nitrate-rich meals were consumed. Maximal urinary excretion was attained 4-6 hours after consumption of a meal and the maximum was proportional to the dose. Plasma nitrate was elevated nine-fold, 1 hour after consumption of a meal containing 128.3 mg nitrate. Plasma nitrated protein levels did not appear to alter significantly from basal 1 hour after supplementation with a nitrate-rich meal. Thus dietary nitrate does not appear to contribute to the levels of plasma nitrated proteins, as determined using a competitive inhibition of binding ELISA assay, but this does not preclude any contribution it may make to the total body burden of nitrotyrosine.
Assuntos
Nitratos/administração & dosagem , Nitritos/administração & dosagem , Tirosina/análogos & derivados , Adulto , Ligação Competitiva , Proteínas Sanguíneas/metabolismo , Dieta , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitratos/sangue , Nitratos/urina , Nitritos/sangue , Nitritos/urina , Sensibilidade e Especificidade , Tirosina/sangue , Tirosina/urinaRESUMO
Exposure of tyrosine or DNA bases to acidic nitrite at low pH results in the nitration of tyrosine and the formation of base deamination products, respectively. At pH 1, hypoxanthine and xanthine are formed from the deamination of adenine and guanine, respectively, whereas under the same conditions, uracil is not detected. The yield of 3-nitrotyrosine derived from interaction of equimolar nitrite and tyrosine at pH 1 is approximately 50% of that obtained from equimolar peroxynitrite-tyrosine interactions at pH 7. 4. The ability of a range of plant phenolic constituents to prevent damage mediated by acidic nitrite was also examined in comparison with the activity of vitamin C. The epicatechin/gallate family of flavonols, constituents of green tea, red wine, etc., demonstrates the most extensive inhibitory properties against both tyrosine nitration and base deamination. The results also show that ascorbic acid is a poor inhibitor of nitration or deamination under acidic conditions such as those of the stomach. The ability of plant phenolics to scavenge reactive nitrogen species derived from acidic nitrite may contribute to the protective effects of tea polyphenols against gastric cancer.