RESUMO
Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a multisystem autoimmune disease with small-vessel involvement. In AAV, microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) are major clinicopathologic variants. In addition, myeloperoxidase (MPO) and proteinase 3 (PR3) are major target antigens. The objective of the study was to explore the predictive factors for long-term survival in AAV patients. Materials and Methods: A multicenter retrospective study was carried out on 407 patients between 2005 and 2020. Clinical parameters were obtained from laboratory tests including the ANCA types, antinuclear antibody (ANA), extractable nuclear antigen (ENA), anti-streptolysin O (ASO), glomerular filtration rate (GFR), and the laboratory examinations for the blood routine, liver function, renal function, and immunity, etc. The data for clinical parameters were collected from electronic medical records (EMRs), and the data for patient survival were acquired through regular follow-up. The association of clinical parameters with overall survival (OS) along with 3-year and 5-year survival rates was analyzed, and the nomogram as a predictive model was established according to the analysis results. Results: In the present study, 336 (82.6%) patients and 46 (11.3%) patients were diagnosed with MPA and GPA, respectively. The mean and median OS for all the patients were 2,285 and 2,290 days, respectively. The 1-year, 3-year, 5-year, and 10-year cumulative survival rates for all the patients were 84.2%, 76.3%, 57.2%, and 32.4%, respectively. Univariate and multivariate survival analyses indicated that the independent prognostic factors included age, pathological categories (MPA, GPA, and other types), serum ANCA types (negative or positive for MPO and/or PR3), ANA, ASO, GFR, lymphocyte, neutrophil-to-lymphocyte ratio (NLR), and C-reactive protein (CRP), and these clinical parameters except for ASO were used to construct a nomogram. The nomogram for 3-year and 5-year survival rates had a C-index of 0.721 (95% CI 0.676-0.766). The calibration curves showed that the predicted values of the nomogram for 3-year and 5-year survival rates were generally consistent with practical observed values, and decision curve analysis (DCA) further demonstrated the practicability and accuracy of the predictive model. Conclusion: Laboratory tests at diagnosis have great significance in the prediction of long-term survival in AAV patients.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Granulomatose com Poliangiite , Poliangiite Microscópica , Anticorpos Anticitoplasma de Neutrófilos , Humanos , Mieloblastina , Prognóstico , Estudos RetrospectivosRESUMO
Although KRAS has long been considered undruggable, direct KRASG12C inhibitors have shown promising initial clinical efficacy. However, the majority of patients still fail to respond. Adaptive feedback reactivation of RAS-mitogen-activated protein kinase (MAPK) signaling has been proposed by our group and others as a key mediator of resistance, but the exact mechanism driving reactivation and the therapeutic implications are unclear. We find that upstream feedback activation of wild-type RAS, as opposed to a shift in KRASG12C to its active guanosine triphosphate (GTP)-bound state, is sufficient to drive RAS-MAPK reactivation in a KRASG12C-independent manner. Moreover, multiple receptor tyrosine kinases (RTKs) can drive feedback reactivation, potentially necessitating targeting of convergent signaling nodes for more universal efficacy. Even in colorectal cancer, where feedback is thought to be primarily epidermal growth factor receptor (EGFR)-mediated, alternative RTKs drive pathway reactivation and limit efficacy, but convergent upstream or downstream signal blockade can enhance activity. Overall, these data provide important mechanistic insight to guide therapeutic strategies targeting KRAS.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Animais , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Retroalimentação , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de SinaisRESUMO
Purpose: To evaluate the antitumor efficacy of cetuximab in combination with LSN3074753, an analog of LY3009120 and pan-RAF inhibitor in 79 colorectal cancer patient-derived xenograft (PDX) models.Experimental Design: Seventy-nine well-characterized colorectal cancer PDX models were employed to conduct a single mouse per treatment group (n = 1) trial.Results: Consistent with clinical results, cetuximab was efficacious in wild-type KRAS and BRAF PDX models, with an overall response rate of 6.3% and disease control rate (DCR) of 20.3%. LSN3074753 was active in a small subset of PDX models that harbored KRAS or BRAF mutations. However, the combination treatment displayed the enhanced antitumor activity with DCR of 35.4%. Statistical analysis revealed that BRAF and KRAS mutations were the best predictors of the combinatorial activity and were significantly associated with synergistic effect with a P value of 0.01 compared with cetuximab alone. In 12 models with BRAF mutations, the combination therapy resulted in a DCR of 41.7%, whereas either monotherapy had a DCR of 8.3%. Among 44 KRAS mutation models, cetuximab or LSN3074753 monotherapy resulted in a DCR of 13.6% or 11.4%, respectively, and the combination therapy increased DCR to 34.1%. Molecular analysis suggests that EGFR activation is a potential feedback and resistant mechanism of pan-RAF inhibition.Conclusions: MAPK and EGFR pathway activations are two major molecular hallmarks of colorectal cancer. This mouse PDX trial recapitulated clinical results of cetuximab. Concurrent EGFR and RAF inhibition demonstrated synergistic antitumor activity for colorectal cancer PDX models with a KRAS or BRAF mutation. Clin Cancer Res; 23(18); 5547-60. ©2017 AACR.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Cetuximab/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Receptores ErbB/metabolismo , Humanos , Ligantes , Camundongos , Compostos de Fenilureia/farmacologia , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirimidinas/farmacologia , Taxa de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Mutação , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Predisposição Genética para Doença , Células HCT116 , Células HT29 , Humanos , Fenótipo , Proteínas Proto-Oncogênicas A-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas A-raf/genética , Proteínas Proto-Oncogênicas A-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Interferência de RNA , Ratos Nus , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacosRESUMO
UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Deleção de Genes , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Expressão Ectópica do Gene , Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/farmacologia , Proteínas ras/genética , Linhagem Celular Tumoral , Dimerização , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação/efeitos dos fármacos , Mutação/genética , Neoplasias/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Isoformas de Proteínas/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/química , Pirimidinas/farmacologia , Proteínas ras/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Linhagem Celular Tumoral/efeitos dos fármacos , Técnicas de Química Sintética , Cães , Feminino , Meia-Vida , Humanos , Masculino , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacocinética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genéticaRESUMO
B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma.
Assuntos
Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Transfecção , Regulação para Cima/efeitos dos fármacos , Vemurafenib , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.
Assuntos
Biologia Computacional , Amplificação de Genes , Genômica , Neoplasias/genética , Oncogenes/genética , Transformação Celular Neoplásica/genética , Mapeamento Cromossômico , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Conjuntos de Dados como Assunto , Epigênese Genética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases , Neoplasias/tratamento farmacológico , Proteínas , Proteínas Proto-Oncogênicas/genéticaRESUMO
Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC(50)) and CETP activity in human plasma (36 nM IC(50)) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED(50) of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development.
Assuntos
Benzodiazepinas/efeitos adversos , Benzodiazepinas/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , HDL-Colesterol/sangue , Aldosterona/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Ratos , Especificidade por SubstratoRESUMO
It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.
Assuntos
Membrana Celular/metabolismo , Resistência à Insulina , Insulina/metabolismo , Esfingomielinas/metabolismo , Animais , Teste de Tolerância a Glucose , Células Hep G2 , Heterozigoto , Humanos , Insulina/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Receptor de Insulina/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingomielinas/análise , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Niacin (nicotinic acid) has recently been shown to increase serum adiponectin concentrations in men with the metabolic syndrome. However, little is known about the mechanism(s) by which niacin regulates the intracellular trafficking and secretion of adiponectin. Since niacin appears to exert its effects on lipolysis through receptor (GPR109A)-dependent and -independent pathways, the purpose of this investigation was to examine the role of the recently identified GPR109A receptor in adiponectin secretion. Initial in vivo studies in rats demonstrated that niacin (30 mg/kg po) acutely increases serum adiponectin concentrations, whereas it decreases NEFAs. Further in vitro studies demonstrated an increase in adiponectin secretion and a decrease in lipolysis in primary adipocytes following treatment with niacin or beta-hydroxybutyrate (an endogenous ligand of the GPR109A receptor), but these effects were blocked when adipocytes were pretreated with pertussis toxin. Niacin had no effect on adiponectin secretion or lipolysis in 3T3-L1 adipocytes, which have limited cell surface expression of the GPR109A receptor. To further substantiate these in vitro findings, wild-type and GPR109A receptor knockout mice were administered a single dose of niacin or placebo, and serum was obtained for the determination of adiponectin and NEFA concentrations. Serum adiponectin concentrations increased and serum NEFAs decreased in the wild-type mice within 10 min following niacin administration. However, niacin administration had no effect on adiponectin and NEFA concentrations in the GPR109A receptor knockout mice. These results demonstrate that the GPR109A receptor plays an important role in the dual regulation of adiponectin secretion and lipolysis.
Assuntos
Hipolipemiantes/farmacologia , Niacina/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adiponectina/sangue , Adiponectina/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Ácidos Graxos não Esterificados/sangue , Humanos , Lipólise/efeitos dos fármacos , Lipólise/fisiologia , Camundongos , Camundongos Knockout , Toxina Pertussis/farmacologia , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genéticaRESUMO
Prebeta1 HDL is the initial plasma acceptor of cell-derived cholesterol in reverse cholesterol transport. Recently, small amphipathic peptides composed of D-amino acids have been shown to mimic apolipoprotein A-I (apoA-I) as a precursor for HDL formation. ApoA-I mimetic peptides have been proposed to stimulate the formation of prebeta1 HDL and increase reverse cholesterol transport in apoE-null mice. The existence of a monoclonal antibody (MAb 55201) and a corresponding ELISA method that is selective for the detection of the prebeta(1) subclass of HDL provides a means of establishing a correlation between apoA-I mimetic dose and prebeta1 HDL formation in human plasma. Using this prebeta1 HDL ELISA, we demonstrate marked apoA-I mimetic dose-dependent prebeta1 HDL formation in human plasma. These results correlated with increases in band density of the plasma prebeta1 HDL, when observed by Western blotting, as a function of increased apoA-I mimetic concentration. Increased prebeta1 HDL formation was observed after as little as 1 min and was maximal within 1 h. Together, these data suggest that a high-throughput prebeta1 HDL ELISA provides a way to quantitatively measure a key component of the reverse cholesterol transport pathway in human plasma, thus providing a possible method for the identification of apoA-I mimetic molecules.
Assuntos
Apolipoproteína A-I , Lipoproteínas de Alta Densidade Pré-beta/biossíntese , Peptídeos/farmacologia , Transporte Biológico , Colesterol/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Lipoproteínas de Alta Densidade Pré-beta/efeitos dos fármacos , Humanos , Cinética , Mimetismo MolecularRESUMO
Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.
Assuntos
Apolipoproteínas E/biossíntese , Colesterol/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Ácido Retinoico/fisiologia , Fatores de Transcrição/fisiologia , Animais , Anticolesterolemiantes/farmacologia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA , Dimerização , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Hidrocarbonetos Fluorados , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nucleares Órfãos , Receptores X de Retinoides , SulfonamidasRESUMO
Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St. John's wort (pregnane X receptor, NR1I2). In this study, we identified the first nonoxysterol natural product that functions as a ligand for the liver X receptor (LXRalpha and LXRbeta; NR1H3, NR1H2), a NHR that acts as the receptor for oxysterols and plays a key role in regulation of cholesterol metabolism and transport as well as glucose metabolism. We show that paxilline, a fungal metabolite, is an efficacious agonist of both LXRalpha and LXRbeta in biochemical and in vitro cell-based assays. Paxilline binds directly to both receptors and is an activator of LXR-dependent transcription in cell-based reporter assays. We also demonstrate that paxilline binding to the receptors results in efficient activation of transcription of two physiological LXR target genes, ABCA1 and SREBP. The discovery of paxilline, the first reported nonoxysterol natural product ligand of the LXRs, may provide insight into the mechanism of ligand recognition by these receptors and reaffirms the utility of examining natural product libraries for identifying novel NHR ligands.
Assuntos
Indóis/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores de Esteroides/agonistas , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Fatores Biológicos/farmacologia , Células Cultivadas , Proteínas de Ligação a DNA , Humanos , Ligantes , Receptores X do Fígado , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , TransfecçãoRESUMO
ABCA1 is an ATP binding cassette transporter that plays an essential role in cholesterol and phospholipid efflux and HDL biogenesis. ABCA1 expression in macrophage cells is subject to regulation by cAMP, cholesterol loading, and ligands of the nuclear receptors liver X receptor (LXR) and retinoid X receptor (RXR). We report here the development of a rapid and high volume branched DNA (bDNA) method to measure ABCA1 mRNA. By using the bDNA method, we show that both LXR and RXR ligands effectively regulate ABCA1 expression in three macrophage cell types: mouse RAW264.7 cell line, mouse peritoneal macrophage cells, and human macrophage THP-1 cells and their regulation is additive. Furthermore, by using a radiolabeled cholesterol efflux assay, we show that both LXR and RXR ligands are sufficient to mediate cholesterol efflux in macrophage cells and their efficacy correlates with ABCA1 regulation. These studies strengthen further the notion that LXR and RXR mediate ABCA1 expression and cholesterol efflux in macrophage cells as a permissive heterodimer and development of small molecule ligands of these nuclear receptors may represent a promising approach to modulating cholesterol efflux and plasma HDL cholesterol level in humans.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Macrófagos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Animais , Sequência de Bases , Transporte Biológico Ativo , Linhagem Celular , Colesterol/metabolismo , Sondas de DNA/genética , Proteínas de Ligação a DNA , Humanos , Ligantes , Receptores X do Fígado , Camundongos , Técnicas de Sonda Molecular , Receptores Nucleares Órfãos , Receptores X de Retinoides , Regulação para CimaRESUMO
Liver X receptors (LXR) belong to the nuclear receptor superfamily that can regulate important lipid metabolic pathways. The plasma phospholipid transfer protein (PLTP) is known to mediate transfer of phospholipids from triglyceride-rich lipoproteins to high density lipoprotein (HDL) and plays a critical role in HDL metabolism. We report here that a specific LXR agonist, T0901317, elevated HDL cholesterol and phospholipid in C57/BL6 mice and generated enlarged HDL particles that were enriched in cholesterol, ApoAI, ApoE, and phospholipid. The appearance of these HDL particles upon oral dosing of T0901317 in C57/BL6 mice was closely correlated with the increased plasma PLTP activity and liver PLTP mRNA levels. Nuclear run-on assay indicated that the effect of LXR agonist on PLTP expression was at the transcriptional level. In mouse peritoneal macrophage cells, PLTP expression was also up-regulated by the LXR/RXR (retinoid X receptor) heterodimer. However, cholesterol efflux in mouse peritoneal macrophage cells from PLTP-deficient mice (PLTP0) was not significantly different from wild type animals. Although in PLTP-deficient mice, the induction of HDL cholesterol as well as HDL particle size increase persisted, the extent of the induction was greatly attenuated. We conclude that PLTP is a direct target gene of LXRs in vivo and plays an important role in LXR agonist-mediated HDL cholesterol and size increase in mice.