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iScience ; 27(2): 108968, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38327788


Excessive or aberrant NLRP3 inflammasome activation has been implicated in the progression and initiation of many inflammatory conditions; however, currently no NLRP3 inflammasome inhibitors have been approved for therapeutic use in the clinic. Here we have identified that the natural product brazilin effectively inhibits both priming and activation of the NLRP3 inflammasome in cultured murine macrophages, a human iPSC microglial cell line and in a mouse model of acute peritoneal inflammation. Through computational modeling, we predict that brazilin can adopt a favorable binding pose within a site of the NLRP3 protein which is essential for its conformational activation. Our results not only encourage further evaluation of brazilin as a therapeutic agent for NLRP3-related inflammatory diseases, but also introduce this small-molecule as a promising scaffold structure for the development of derivative NLRP3 inhibitor compounds.

J Biol Chem ; 298(4): 101777, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35231445


Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin-TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin-TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin-TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.

Proteínas de Transporte , Quinase 1 do Ponto de Checagem , Estresse Fisiológico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem/metabolismo , Replicação do DNA/fisiologia , Fosforilação
Cancer Res ; 82(2): 307-319, 2022 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34810199


PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Antineoplásicos/administração & dosagem , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/metabolismo , Dano ao DNA/efeitos dos fármacos , Reposicionamento de Medicamentos/métodos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Sulfonas/administração & dosagem , Animais , Carcinoma Epitelial do Ovário/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos SCID , Neoplasias Ovarianas/patologia , Células PC-3 , Reparo de DNA por Recombinação/efeitos dos fármacos , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
NAR Cancer ; 3(3): zcab028, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34316715


Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Sci Rep ; 9(1): 3617, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837643


CPX-351 is a liposomally encapsulated 5:1 molar ratio of cytarabine and daunorubicin that recently received regulatory approval for the treatment of therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes based on improved overall survival compared to standard cytarabine/daunorubicin therapy. Checkpoint kinase 1 (CHK1), which is activated by DNA damage and replication stress, diminishes sensitivity to cytarabine and anthracyclines as single agents, suggesting that CHK1 inhibitors might increase the effectiveness of CPX-351. The present studies show that CPX-351 activates CHK1 as well as the S and G2/M cell cycle checkpoints. Conversely, CHK1 inhibition diminishes the cell cycle effects of CPX-351. Moreover, CHK1 knockdown or addition of a CHK1 inhibitor such as MK-8776, rabusertib or prexasertib enhances CPX-351-induced apoptosis in multiple TP53-null and TP53-wildtype AML cell lines. Likewise, CHK1 inhibition increases the antiproliferative effect of CPX-351 on primary AML specimens ex vivo, offering the possibility that CPX-351 may be well suited to combine with CHK1-targeted agents.

Apoptose , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Citarabina/farmacologia , Daunorrubicina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/patologia , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células , Humanos , Técnicas In Vitro , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Células Tumorais Cultivadas
Transplantation ; 103(6): 1253-1259, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30335695


BACKGROUND: Although exercise capacity improves postheart transplantation (HTx), it remains unclear if the level of physical activity (PA) shows similar improvement. The purpose of this study was to (1) describe PA levels and (2) identify factors which may be associated with levels of PA post-HTx. METHODS: A prospective observational cross-sectional study was conducted at a single center HTx outpatient clinic. Medically stable adult recipients 6 months or longer post-HTx were recruited. Physical activity level (PAL) and average daily time spent at least moderately active (≥3 metabolic equivalents) were estimated using a multisensor device. Factors investigated were demographic (age, sex, body mass index [BMI], time post-HTx, and reason for HTx), corticosteroid use, exercise capacity (6-min walk distance), and quadriceps muscle strength corrected for body weight (QS%). RESULTS: The mean post-HTx time of the 75 participants was 9.2 ± 7.0 years (0.5-26 y). Twenty-seven (36%) participants were classified as extremely inactive (PAL, <1.40), 26 (34.6%) sedentary (1.40 ≤ PAL ≤ 1.69), and 22 (29.3%) active (PAL, ≥1.70). Multivariable analysis showed greater QS% (ß = 0.004 (0.002-0.006) P = 0.001) to be independently associated with increased PAL. For increased time, 3 or more metabolic equivalents both greater QS% (ß = 0.0164 [0.003-0.029]; P = 0.014) and lower BMI (ß = -0.0626 [-0.115 to -0.0099]; P = 0.021) were independently associated. CONCLUSIONS: The degree of observed sedentary behavior post-HTx is surprising, with the majority of participants not reaching levels of PA recommended for health benefits. QS% and BMI were the only factors found to be independently associated with estimates of PA. Further quality trials are required to demonstrate the long-term benefits of regular PA and investigate ways of increasing adherence to PA post-HTx.

Índice de Massa Corporal , Tolerância ao Exercício , Exercício Físico , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Força Muscular , Músculo Quadríceps/fisiopatologia , Comportamento Sedentário , Adulto , Idoso , Estudos Transversais , Feminino , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/fisiopatologia , Transplante de Coração/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Aptidão Física , Estudos Prospectivos , Recuperação de Função Fisiológica , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento