RESUMO
Fluorescence lifetime imaging microscopy of a fluorescence probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), provides an objective method for preoperative diagnosis of fine-needle aspiration (FNA) of thyroid nodules. The key of this o-BMVC test of FNA smears is the measurement of the digital number of o-BMVC foci in the nucleus. Thus, there are three categories classified in the o-BMVC test, which are nondiagnostic for unsatisfactory samples, benign for less numbers of o-BMVC foci, and malignant for more numbers of o-BMVC foci. The discrimination of indeterminate (including atypia, follicular neoplasm, suspicious) cytology into benign or malignant cases can reduce diagnostic uncertainty and benefit clinical decision making. This pilot study strongly suggests that the o-BMVC test is an invaluable method for diagnosing FNA samples. Particularly, the combination of FNA cytology and the o-BMVC test holds great promise to improve the efficacy of diagnosis and reduce the healthcare costs.
Assuntos
Nódulo da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina/métodos , Carbazóis/administração & dosagem , Citodiagnóstico/métodos , Feminino , Corantes Fluorescentes/administração & dosagem , Humanos , Masculino , Microscopia de Fluorescência/métodos , Pessoa de Meia-Idade , Projetos Piloto , Compostos de Piridínio/administração & dosagem , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/patologia , Adulto JovemRESUMO
Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss-Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Glicosídeo Hidrolases/metabolismo , Niacinamida/análogos & derivados , Compostos de Piridínio/farmacocinética , Células A549 , ADP-Ribosil Ciclase/genética , Administração Oral , Envelhecimento/efeitos dos fármacos , Animais , Antígenos CD/genética , Suplementos Nutricionais , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Microbioma Gastrointestinal , Glicosídeo Hidrolases/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Camundongos , Camundongos Knockout , Niacina/metabolismo , Niacinamida/administração & dosagem , Niacinamida/metabolismo , Niacinamida/farmacocinética , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Compostos de Piridínio/administração & dosagemRESUMO
Acute kidney injury (AKI) is associated with a very high mortality and an increased risk for progression to chronic kidney disease (CKD). Ischemia-reperfusion injury (IRI) is a model for AKI, which results in tubular damage, dysfunction of the mitochondria and autophagy, and in decreased cellular nicotinamide adenine dinucleotide (NAD+) with progressing fibrosis resulting in CKD. NAD+ is a co-enzyme for several proteins, including the NAD+ dependent sirtuins. NAD+ augmentation, e.g. by use of its precursor nicotinamide riboside (NR), improves mitochondrial homeostasis and organismal metabolism in many species. In the present investigation the effects of prophylactic administration of NR on IRI-induced AKI were studied in the rat. Bilateral IRI reduced kidney tissue NAD+, caused tubular damage, reduced α-Klotho (klotho), and altered autophagy flux. AKI initiated progression to CKD, as shown by induced profibrotic Periostin (postn) and Inhibin subunit beta-A, (activin A / Inhba), both 24 hours and 14 days after surgery. NR restored tissue NAD+ to that of the sham group, increased autophagy (reduced p62) and sirtuin1 (Sirt1) but did not ameliorate renal tubular damage and profibrotic genes in the 24 hours and 14 days IRI models. AKI induced NAD+ depletion and impaired autophagy, while augmentation of NAD+ by NR restored tissue NAD+ and increased autophagy, possibly serving as a protective response. However, prophylactic administration of NR did not ameliorate tubular damage of the IRI rats nor rescued the initiation of fibrosis in the long-term AKI to CKD model, which is a pivotal event in CKD pathogenesis.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , NAD/metabolismo , Niacinamida/análogos & derivados , Substâncias Protetoras/administração & dosagem , Compostos de Piridínio/administração & dosagem , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Injúria Renal Aguda/complicações , Injúria Renal Aguda/metabolismo , Animais , Autofagia/efeitos dos fármacos , Progressão da Doença , Fibrose , Glucuronidase/metabolismo , Rim/metabolismo , Rim/patologia , Proteínas Klotho , Masculino , Mitocôndrias/metabolismo , Niacinamida/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Wistar , Insuficiência Renal Crônica/etiologia , Sirtuína 1/metabolismo , Resultado do TratamentoRESUMO
Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A-T). Loss of mitochondrial function can drive age-related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence-associated secretory phenotype (SASP) occur in A-T patient fibroblasts, and in ATM-deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD+ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1-dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm-/- mice. Our findings suggest a central role for mitochondrial dysfunction-induced senescence in A-T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.
Assuntos
Ataxia Telangiectasia/dietoterapia , Ataxia Telangiectasia/metabolismo , Suplementos Nutricionais , Proteínas de Membrana/metabolismo , Mitofagia/efeitos dos fármacos , NAD/metabolismo , Niacinamida/análogos & derivados , Compostos de Piridínio/administração & dosagem , Fenótipo Secretor Associado à Senescência/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitofagia/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Niacinamida/administração & dosagem , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transfecção , Resultado do TratamentoRESUMO
Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Óxidos N-Cíclicos/farmacologia , Indolizinas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Compostos de Piridínio/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Óxidos N-Cíclicos/administração & dosagem , Sinergismo Farmacológico , Feminino , Amplificação de Genes , Humanos , Indóis/administração & dosagem , Indóis/farmacologia , Indolizinas/administração & dosagem , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Oxazóis/administração & dosagem , Oxazóis/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , Compostos de Piridínio/administração & dosagem , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Distribuição Aleatória , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Hypoxia-inducible factor (HIF) is one of the critical components of the tumor microenvironment that is involved in tumor development. HIF-1α functionally and physically interacts with CDK1, 2, and 5 and stimulates the cell cycle progression and Cyclin-Dependent Kinase (CDK) expression. Therefore, hypoxic tumor microenvironment and CDK overexpression lead to increased cell cycle progression and tumor expansion. Therefore, we decided to suppress cancer cell expansion by blocking HIF-1α and CDK molecules. METHODS: In the present study, we used the carboxylated graphene oxide (CGO) conjugated with trimethyl chitosan (TMC) and hyaluronate (HA) nanoparticles (NPs) loaded with HIF-1α-siRNA and Dinaciclib, the CDK inhibitor, for silencing HIF-1α and blockade of CDKs in CD44-expressing cancer cells and evaluated the impact of combination therapy on proliferation, metastasis, apoptosis, and tumor growth. RESULTS: The results indicated that the manufactured NPs had conceivable physicochemical properties, high cellular uptake, and low toxicity. Moreover, combination therapy of cancer cells using CGO-TMC-HA NPs loaded with HIF-1α siRNA and Dinaciclib (SCH 727965) significantly suppressed the CDKs/HIF-1α and consequently, decreased the proliferation, migration, angiogenesis, and colony formation in tumor cells. CONCLUSIONS: These results indicate the ability of CGO-TMC-HA NPs for dual drug/gene delivery in cancer treatment. Furthermore, the simultaneous inhibition of CDKs/HIF-1α can be considered as a novel anti-cancer treatment strategy; however, further research is needed to confirm this treatment in vivo. Graphical Abstract The suppression of HIF-1α and CDKs inhibits cancer growth. HIF-1α is overexpressed by the cells present in the tumor microenvironment. The hypoxic environment elevates mitochondrial ROS production and increases p38 MAP kinase, JAK/STAT, ERK, JNK, and Akt/PI3K signaling, resulting in cyclin accumulation and aberrant cell cycle progression. Furthermore, the overexpression of HIF-1α/CDK results in increased expression of genes such as BCL2, Bcl-xl, Ki-67, TGFß, VEGF, FGF, MMP2, MMP9, and, HIF-1α and consequently raise the survival, proliferation, angiogenesis, metastasis, and invasion of tumor cells. In conclusion, HIF-1α-siRNA/Dinaciclib-loaded CGO-TMC-HA NPs can inhibit the tumor expansion by blockage of CDKs and HIF-1α (JAK: Janus kinase, STAT: Signal transducer and activator of transcription, MAPK: mitogen-activated protein kinase, ERK: extracellular signal-regulated kinase, JNK: c-Jun N-terminal kinase, PI3K: phosphatidylinositol 3-kinase).
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Experimentais/terapia , Compostos de Piridínio/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Quitosana/química , Óxidos N-Cíclicos , Grafite/química , Ácido Hialurônico/química , Indolizinas , Camundongos , Nanopartículas/química , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Compostos de Piridínio/química , Compostos de Piridínio/farmacocinética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinéticaRESUMO
The combination of drugs targeting Ral and PI3K/AKT signaling has antitumor efficacy in preclinical models of pancreatic cancer. We combined dinaciclib (small molecule cyclin dependent kinase inhibitor with MK-2206 (Akt inhibitor) in patients with previously treated/metastatic pancreatic cancer. Patients were treated with dinaciclib (6-12 mg/m2 i.v.) and MK-2206 (60-135 mg p.o.) weekly. Tumor biopsies were performed to measure pAKT, pERK, and Ki67 at baseline and after one completed cycle (dose level 2 and beyond). Thirty-nine patients participated in the study. The maximum tolerated doses were dinaciclib 9 mg/m2 and MK-2206 135 mg. Treatment-related grade 3 and 4 toxicities included neutropenia, lymphopenia, anemia, hyperglycemia, hyponatremia, and leukopenia. No objectives responses were observed. Four patients (10%) had stable disease as their best response. At the recommended dose, median survival was 2.2 months. Survival rates at 6 and 12 months were 11% and 5%, respectively. There was a nonsignificant reduction in pAKT composite scores between pretreatment and post-treatment biopsies (mean 0.76 vs. 0.63; P = 0.635). The combination of dinaciclib and MK-2206 was a safe regimen in patients with metastatic pancreatic cancer, although without clinical benefit, possibly due to not attaining biologically effective doses. Given the strong preclinical evidence of Ral and AKT inhibition, further studies with better tolerated agents should be considered.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Carcinoma Ductal Pancreático/tratamento farmacológico , Óxidos N-Cíclicos/toxicidade , Compostos Heterocíclicos com 3 Anéis/toxicidade , Indolizinas/toxicidade , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/toxicidade , Compostos de Piridínio/toxicidade , Administração Oral , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biópsia , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Óxidos N-Cíclicos/administração & dosagem , Feminino , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Humanos , Indolizinas/administração & dosagem , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Compostos de Piridínio/administração & dosagem , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Oxime-based acetylcholinesterase reactivators (briefly oximes) regenerate organophosphate-inactivated acetylcholinesterase and restore its function. Poor blood-brain-barrier passage and fast elimination from blood limit their actual use in treatment of patients exposed to organophosphates. Previous in vitro results implicated further testing of cucurbit[7]uril as a delivery vehicle for bisquaternary oximes. The present paper focuses on cell toxicity, in vivo safety and influence of cucurbit[7]uril on oxime pharmacokinetics and pharmacodynamics. Neither the K027 nor the complex caused any cell toxicity, changes in blood biochemistry or hepato- or nephrotoxicity in tested concentrations. The encapsulation of K027 increased and accelerated the blood-brain-barrier penetration. The peripheral oxime exposure also increased, supporting the suggestion that cucurbit[7]uril protects the circulating oxime from rapid renal clearance. Contrary to the comparable in vitro reactivation power of K027 and the encapsulated K027, we failed to confirm this in vivo. In theory, this might result from the non-specific binding of molecules to the cucurbit[7]uril or the interaction of K027 with cucurbit[7]uril being too strong for acetylcholinesterase reactivation. Precise explanation requires additional in silico, in vitro and also in vivo experiments.
Assuntos
Acetilcolinesterase/sangue , Acetilcolinesterase/metabolismo , Encéfalo/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Reativadores da Colinesterase/farmacocinética , Eritrócitos/efeitos dos fármacos , Imidazóis/farmacocinética , Oximas/farmacocinética , Compostos de Piridínio/farmacocinética , Células A549 , Animais , Encéfalo/enzimologia , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Hidrocarbonetos Aromáticos com Pontes/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Reativadores da Colinesterase/administração & dosagem , Reativadores da Colinesterase/toxicidade , Relação Dose-Resposta a Droga , Eritrócitos/enzimologia , Feminino , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/metabolismo , Células Hep G2 , Humanos , Imidazóis/administração & dosagem , Imidazóis/toxicidade , Injeções Intramusculares , Masculino , Dose Máxima Tolerável , Camundongos Endogâmicos ICR , Oximas/administração & dosagem , Oximas/toxicidade , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/toxicidade , Medição de Risco , Distribuição TecidualRESUMO
Tumor-associated myeloid cells (TAMCs) are key drivers of immunosuppression in the tumor microenvironment, which profoundly impedes the clinical response to immune-dependent and conventional therapeutic modalities. As a hallmark of glioblastoma (GBM), TAMCs are massively recruited to reach up to 50% of the brain tumor mass. Therefore, they have recently been recognized as an appealing therapeutic target to blunt immunosuppression in GBM with the hope of maximizing the clinical outcome of antitumor therapies. Here we report a nano-immunotherapy approach capable of actively targeting TAMCs in vivo. As we found that programmed death-ligand 1 (PD-L1) is highly expressed on glioma-associated TAMCs, we rationally designed a lipid nanoparticle (LNP) formulation surface-functionalized with an anti-PD-L1 therapeutic antibody (αPD-L1). We demonstrated that this system (αPD-L1-LNP) enabled effective and specific delivery of therapeutic payload to TAMCs. Specifically, encapsulation of dinaciclib, a cyclin-dependent kinase inhibitor, into PD-L1-targeted LNPs led to a robust depletion of TAMCs and an attenuation of their immunosuppressive functions. Importantly, the delivery efficiency of PD-L1-targeted LNPs was robustly enhanced in the context of radiation therapy (RT) owing to the RT-induced up-regulation of PD-L1 on glioma-infiltrating TAMCs. Accordingly, RT combined with our nano-immunotherapy led to dramatically extended survival of mice in 2 syngeneic glioma models, GL261 and CT2A. The high targeting efficiency of αPD-L1-LNP to human TAMCs from GBM patients further validated the clinical relevance. Thus, this study establishes a therapeutic approach with immense potential to improve the clinical response in the treatment of GBM and warrants a rapid translation into clinical practice.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Células Mieloides/patologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Óxidos N-Cíclicos , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Humanos , Indolizinas , Camundongos , Células Mieloides/efeitos dos fármacos , Células Mieloides/efeitos da radiação , Nanopartículas , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/uso terapêutico , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The major function of compounds with an oxime moiety attached to a quarternary nitrogen pyridinium ring is to reactivate acetylcholinesterase inhibited by organophosphorus agent (OP). However, other oxime mechanisms (e.g. modulation of cholinergic or glutamatergic receptor) may be involved in the recovery. The main disadvantage of positively charged reactivators is their low ability to penetrate into the brain although crossing the blood brain barrier could be supported via increasing the dose of administered oxime. Thus, this study presents maximal tolerated doses (MTD) for marketed oximes (TMB-4, MMB-4, LüH-6, HI-6, 2-PAM) and the most promising K-oximes (K027, K048, K203) which can be used in OP therapy in the future. No signs of sarin intoxication were observed in mice treated with 100% MTD of HI-6 in contrast to those treated with atropine and only 5% LD50 of HI-6. 100% MTD of HI-6 resulted in levels of 500⯵M and 12⯵M in plasma and brain, respectively. This concentration is by a far margin safe with respect to direct effects on neuronal cell viability and, on the other hand, does not have any effects on central NMDA receptors or central nACh receptors. However, a weak antimuscarinic activity in case of LüH-6 and a weak peripheral antinicotinic action in case of TMB-4 and 2-PAM could be observed at their respective 100% MTD dose. These high doses, represented by MTD, are, however, irrelevant to clinical practice since they led to mild to moderate toxic side effects. Therefore, we conclude that clinically used doses of marketed oxime reactivators have no significant direct pharmacological effect on the tested receptors.
Assuntos
Reativadores da Colinesterase/administração & dosagem , Dose Máxima Tolerável , Compostos Organofosforados/toxicidade , Oximas/administração & dosagem , Compostos de Piridínio/administração & dosagem , Animais , Células CHO , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Galinhas , Reativadores da Colinesterase/toxicidade , Cricetinae , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oximas/toxicidade , Compostos de Pralidoxima/administração & dosagem , Compostos de Pralidoxima/toxicidade , Compostos de Piridínio/toxicidadeRESUMO
PURPOSE: In this study, we addressed the underlying mechanisms for the association between enzalutamide (ENZ) treatment and neuroendocrine prostate cancer (NEPC), and the critical involvement of MYCN, and loss of RB1 function in neuroendocrine differentiation (NED) of prostatic epithelial cells, and the development of NEPC. We further sought to determine whether PARP inhibition could suppress NEPC, and to identify molecular determinants of this therapeutic activity. EXPERIMENTAL DESIGN: We used a novel prostate cancer patient-derived xenograft (PDX) treatment model, prostatic adenocarcinoma and NEPC cell lines, an NEPC organoid line, and NEPC xenograft models to address the mechanistic basis of ENZ-induced NED, and to analyze suppression of NED and NEPC growth by PARP inhibition. RESULTS: We identified an ENZ treatment-associated glucocorticoid receptor (GR)-MYCN-CDK5-RB1-E2F1 signaling pathway that drives NED in prostatic adenocarcinoma PDX and cell line models. Mechanistically, long-term ENZ treatment transcriptionally upregulates signaling of the GR-MYCN axis, leading to CDK5R1 and CDK5R2 upregulation, Rb1 phosphorylation, and N-Myc-mediated and E2F1-mediated NED gene expression. Importantly, olaparib (OLA) or talazoparib (TALA) suppressed these activities, and the combination of OLA and dinaciclib (DINA), an inhibitor of CDK2 and CDK5, which also inhibits Rb1 phosphorylation, suppressed NED and significantly improved therapeutic efficiency in NEPC cells in vitro and in NEPC tumors in vivo. CONCLUSIONS: The results of our study indicate an important role of GR-MYCN-CDK5R1/2-RB1-NED signaling in ENZ-induced and PARP inhibitor-suppressed NEPC. We also demonstrated efficacy for OLA+DINA combination therapy in NEPC xenograft models.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Tumores Neuroendócrinos/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Proteínas/genética , Animais , Benzamidas , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Linhagem Celular Tumoral , Óxidos N-Cíclicos , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indolizinas , Masculino , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Nitrilas , Feniltioidantoína/administração & dosagem , Feniltioidantoína/análogos & derivados , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Proteínas/metabolismo , Compostos de Piridínio/administração & dosagem , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Resultado do Tratamento , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
OBJECTIVE: This study aimed to identify and evaluate the mechanism by which apoptosis and cell cycle arrest were induced by dinaciclib in lymphoma Raji cells. METHODS: The colony formation assay was used to detect cell proliferation of Raji cells. Cell cycle arrest and cell apoptosis were determined by flow cytometry and TUNEL assays, respectively. Protein expression related to the Raji cell state was evaluated by Western blot. The Raji/Dinaciclib drug-resistant cell line was established, where the regulating functions of CDK1-involved pathway were verified. In addition, the effect of dinaciclib in vivo was examined in orthotopically implanted tumors in nude mice. RESULTS: Cell apoptosis was induced, and DNA synthesis ability was decreased in a time-dependent manner in dinaciclib-treated lymphoma Raji cells. Furthermore, the cell cycle was found to be blocked in the G2/M Phase. Further study indicated that CDK1-involved pathway played a key regulatory role in this process. It was revealed by cell transfection that the expression of cell cycle proteins was downregulated after treatment with dinaciclib through a CDK1-involved pathway, which eventually led to apoptosis. Knockdown of CDK1 restored the sensitivity of the Raji/Dinaciclib cells to dinaciclib. Xenograft model of nude mice showed that dinaciclib treatment in vivo could effectively inhibit tumor growth, consistent with the experiment results mentioned before. CONCLUSION: In this study, we clarified the mechanisms through which dinaciclib induces Raji cell apoptosis and blocks the cell cycle through a CDK1-involved pathway, which supported that dinaciclib had potential values in the treatment of lymphoma.
Assuntos
Antineoplásicos/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Proteína Quinase CDC2/metabolismo , Linfoma/tratamento farmacológico , Compostos de Piridínio/administração & dosagem , Animais , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Indolizinas , Linfoma/metabolismo , Camundongos , Camundongos Nus , Compostos de Piridínio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dinaciclib is a multi-specific cyclin-dependent kinase (CDK) inhibitor with significant preclinical and clinical activity. It inhibits CDK1, CDK2, CDK5, CDK9 and CDK12 in the nanomolar range and exhibits potent antiproliferative effects on various cancers in vitro and in vivo. Aldo-keto reductases (AKR) and carbonyl reductases (CBR) are enzymes involved at the biosynthesis, intermediary metabolism and detoxification processes, but can also play a significant role in cancer resistance. Here, we report that dinaciclib is a strong inhibitor of aldo-keto reductase 1C3 (AKR1C3), an enzyme that is known to be an important regulator of cell proliferation and differentiation. AKR1C3 is overexpressed in a range of cancer types and is also involved in tumour cell resistance to anthracyclines. In our study, dinaciclib displayed tight-binding inhibition of human recombinant AKR1C3 (Kiapp = 0.07 µM) and was also active at the cellular level (IC50 = 0.23 µM). Dinaciclib acts as a noncompetitive inhibitor with respect to daunorubicin and as an uncompetitive inhibitor with respect to the NADPH. In subsequent experiments, pretreatment with dinaciclib (0.1 µM) significantly sensitized AKR1C3-overexpressing anthracycline-resistant cancer cells to daunorubicin. In conclusion, our results indicate that dinaciclib may potentially increase the therapeutic efficacy and safety of anthracyclines by preventing anthracycline resistance and minimizing their adverse effects.
Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos de Piridínio/farmacologia , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Antraciclinas/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Óxidos N-Cíclicos , Daunorrubicina/metabolismo , Daunorrubicina/farmacocinética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células Hep G2 , Humanos , Indolizinas , Compostos de Piridínio/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The BRAFV600E mutation occurs in approximately 8% of human colorectal cancers and is associated with therapeutic resistance that is due, in part, to reactivation of MEK/ERK signaling cascade. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E colorectal cancers. Therefore, it was determined whether CDK1 antagonism enhances the efficacy of MEK inhibition in BRAFV600E colorectal cancer cells. BRAFV600E colorectal cancer cell lines expressing CDK1 were sensitized to apoptosis upon siRNA knockdown or small-molecule inhibition with RO-3306 (CDK1 inhibitor) or dinaciclib (CDK1, 2, 5, 9 inhibitors). Combination of RO-3306 or dinaciclib with cobimetinib (MEK inhibitor) cooperatively enhanced apoptosis and reduced clonogenic survival versus monotherapy. Cells isogenic or ectopic for BRAFV600E displayed resistance to CDK1 inhibitors, as did cells with ectopic expression of constitutively active MEK CDK1 inhibitors induced a CASP8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced CASP3 cleavage. CDK inhibitors suppressed pro-CASP8 phosphorylation at S387, as shown by drug withdrawal, which restored p-S387 and increased mitosis. In a colorectal cancer xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis versus either drug alone. The Cancer Genome Atlas (TCGA) transcriptomic dataset revealed overexpression of CDK1 in human colorectal cancers versus normal colon. Together, these data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose combined targeting with a MEK/ERK inhibitor represents an effective therapeutic strategy.Implications: CDK1 is a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose dual targeting with a MEK inhibitor may be therapeutically effective. Mol Cancer Res; 16(3); 378-89. ©2017 AACR.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Azetidinas/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Proteína Quinase CDC2/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Óxidos N-Cíclicos , Células HT29 , Humanos , Indolizinas , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Mutação , Piperidinas/administração & dosagem , Compostos de Piridínio/administração & dosagem , Quinolinas , Tiazóis , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Anticorpos Monoclonais/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Compostos de Piridínio/administração & dosagem , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Óxidos N-Cíclicos , Feminino , Humanos , Indolizinas , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Compostos de Piridínio/efeitos adversos , RecidivaRESUMO
Purpose: Cyclin E1 (CCNE1) amplification is associated with primary treatment resistance and poor outcome in high-grade serous ovarian cancer (HGSC). Here, we explore approaches to target CCNE1-amplified cancers and potential strategies to overcome resistance to targeted agents.Experimental Design: To examine dependency on CDK2 in CCNE1-amplified HGSC, we utilized siRNA and conditional shRNA gene suppression, and chemical inhibition using dinaciclib, a small-molecule CDK2 inhibitor. High-throughput compound screening was used to identify selective synergistic drug combinations, as well as combinations that may overcome drug resistance. An observed relationship between CCNE1 and the AKT pathway was further explored in genomic data from primary tumors, and functional studies in fallopian tube secretory cells.Results: We validate CDK2 as a therapeutic target by demonstrating selective sensitivity to gene suppression. However, we found that dinaciclib did not trigger amplicon-dependent sensitivity in a panel of HGSC cell lines. A high-throughput compound screen identified synergistic combinations in CCNE1-amplified HGSC, including dinaciclib and AKT inhibitors. Analysis of genomic data from TCGA demonstrated coamplification of CCNE1 and AKT2 Overexpression of Cyclin E1 and AKT isoforms, in addition to mutant TP53, imparted malignant characteristics in untransformed fallopian tube secretory cells, the dominant site of origin of HGSC.Conclusions: These findings suggest a specific dependency of CCNE1-amplified tumors for AKT activity, and point to a novel combination of dinaciclib and AKT inhibitors that may selectively target patients with CCNE1-amplified HGSC. Clin Cancer Res; 23(7); 1862-74. ©2016 AACR.
Assuntos
Ciclina E/genética , Quinase 2 Dependente de Ciclina/genética , Proteína Oncogênica v-akt/genética , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Linhagem Celular Tumoral , Óxidos N-Cíclicos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indolizinas , Proteína Oncogênica v-akt/antagonistas & inibidores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Compostos de Piridínio/administração & dosagem , RNA Interferente Pequeno/genéticaRESUMO
Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is responsible for approximately 15% of cancer-related mortality in children. Aberrant activation of cyclin-dependent kinases (CDKs) has been shown to contribute to tumor cell progression in many cancers including NB. Therefore, small molecule inhibitors of CDKs comprise a strategic option in cancer therapy. Here we show that a novel multiple-CDK inhibitor, dinaciclib (SCH727965, MK-7965), exhibits potent anti-proliferative effects on a panel of NB cell lines by blocking the activity of CDK2 and CDK9. Dinaciclib also significantly sensitized NB cell lines to the treatment of chemotherapeutic agents such as doxorubicin (Dox) and etoposide (VP-16). Furthermore, dinaciclib revealed in vivo antitumor efficacy in an orthotopic xenograft mouse model of two NB cell lines and blocked tumor development in the TH-MYCN transgenic NB mouse model. Taken together, this study suggests that CDK2 and CDK9 are potential therapeutic targets in NB and that abrogating CDK2 and CDK9 activity by small molecules like dinaciclib is a promising strategy and a treatment option for NB patients.
Assuntos
Quinase 2 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/genética , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Óxidos N-Cíclicos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Indolizinas , Camundongos , Neuroblastoma/genética , Neuroblastoma/patologia , Fosforilação/efeitos dos fármacos , Compostos de Piridínio/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: 1-methylnicotinamide (1-MNA), an endogenous metabolite of nicotinamide, has recently gained interest due to its anti-inflammatory and anti-thrombotic activities linked to the COX-2/PGI2 pathway. Given the previously reported anti-metastatic activity of prostacyclin (PGI2), we aimed to assess the effects of 1-MNA and its structurally related analog, 1,4-dimethylpyridine (1,4-DMP), in the prevention of cancer metastasis. METHODS: All the studies on the anti-tumor and anti-metastatic activity of 1-MNA and 1,4-DMP were conducted using the model of murine mammary gland cancer (4T1) transplanted either orthotopically or intravenously into female BALB/c mouse. Additionally, the effect of the investigated molecules on cancer cell-induced angiogenesis was estimated using the matrigel plug assay utilizing 4T1 cells as a source of pro-angiogenic factors. RESULTS: Neither 1-MNA nor 1,4-DMP, when given in a monotherapy of metastatic cancer, influenced the growth of 4T1 primary tumors transplanted orthotopically; however, both compounds tended to inhibit 4T1 metastases formation in lungs of mice that were orthotopically or intravenously inoculated with 4T1 or 4T1-luc2-tdTomato cells, respectively. Additionally, while 1-MNA enhanced tumor vasculature formation and markedly increased PGI2 generation, 1,4-DMP did not have such an effect. The anti-metastatic activity of 1-MNA and 1,4-DMP was further confirmed when both agents were applied with a cytostatic drug in a combined treatment of 4T1 murine mammary gland cancer what resulted in up to 80 % diminution of lung metastases formation. CONCLUSIONS: The results of the studies presented below indicate that 1-MNA and its structural analog 1,4-DMP prevent metastasis and might be beneficially implemented into the treatment of metastatic breast cancer to ensure a comprehensive strategy of metastasis control.
Assuntos
Neoplasias Pulmonares/prevenção & controle , Neoplasias Mamárias Experimentais/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Piridínio/administração & dosagem , Animais , Caderinas/metabolismo , Proteínas Cdh1/metabolismo , Linhagem Celular Tumoral , Epoprostenol , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/irrigação sanguínea , Camundongos , Camundongos Endogâmicos BALB C , Niacinamida/administração & dosagem , Niacinamida/farmacologia , Compostos de Piridínio/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PARP1/2 are required for single-strand break repair, and their inhibition causes DNA replication fork collapse and double-strand break (DSB) formation. These DSBs are primarily repaired via homologous recombination (HR), a high-fidelity repair pathway. Should HR be deficient, DSBs may be repaired via error-prone nonhomologous end-joining mechanisms, or may persist, ultimately resulting in cell death. The combined disruption of PARP and HR activities thus produces synthetic lethality. Multiple myeloma cells are characterized by chromosomal instability and pervasive DNA damage, implicating aberrant DNA repair. Cyclin-dependent kinases (CDK), upstream modulators of HR, are dysregulated in multiple myeloma. Here, we show that a CDK inhibitor, dinaciclib, impairs HR repair and sensitizes multiple myeloma cells to the PARP1/2 inhibitor ABT-888. Dinaciclib abolishes ABT-888-induced BRCA1 and RAD51 foci and potentiates DNA damage, indicated by increased γH2AX foci. Dinaciclib treatment reduces expression of HR repair genes, including Rad51, and blocks BRCA1 phosphorylation, a modification required for HR repair, thus inhibiting HR repair of chromosome DSBs. Cotreatment with dinaciclib and ABT-888 in vitro resulted in synthetic lethality of multiple myeloma cells, but not normal CD19(+) B cells, and slowed growth of multiple myeloma xenografts in SCID mice almost two-fold. These findings support combining dinaciclib with PARP inhibitors for multiple myeloma therapy. Mol Cancer Ther; 15(2); 241-50. ©2015 AACR.
Assuntos
Benzimidazóis/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Quinases Ciclina-Dependentes/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Compostos de Piridínio/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Benzimidazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indolizinas , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Compostos de Piridínio/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: High-grade serous ovarian cancer (HGS-OvCa) is characterized by widespread CCNE1 amplification. Current treatments lack specificity to target Cyclin E-driven OvCa. METHODS: By in silico analysis of the TCGA OvCa dataset we searched association between genes involved in glucose metabolism and cell cycle control. Metabolic shift was studied in Cyclin E-driven OvCa cells treated with CDK inhibition (CDKi). Genetic and pharmaceutical inhibition of succinate dehydrogenase (SDH) was tested in combination with CDKi. RESULTS: OvCa patients with CCNE1 amplification could be divided by concomitant SDHA amplification. A2780 OvCa cells were similar to the Cyclin E-driven and SDHA neutral genotype. CDKi in A2780 cells using Dinaciclib resulted in compensatory enhancement of tricarboxylicacid cycle (TCA) cycle activity. Combined blockade of CDK and SDH, both genetically and pharmaceutically, showed synergy and resulted in inhibited proliferation, migration, invasion and migration in A2780 cells. The combined inhibition did not further alter cell cycle population, but induced apoptosis of A2780 cells. CONCLUSION: Cyclin E-driven OvCa cells appeared addicted to glucose metabolism via TCA. Combined CDKi with modalities targeting TCA, like SDHA inhibition showed promising effects for this genotype.