RESUMO
Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-ß-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and ß-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Quinase 1 de Adesão Focal/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Animais , Cisplatino/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Células-TroncoRESUMO
Mast cell lipid bodies are key to initiation, maintenance and resolution of inflammatory responses in tissue. Mast cell lines, primary bone marrow-derived mast cells and peripheral blood basophils present a 'steatotic' phenotype in response to chronic insulin exposure, where cells become loaded with lipid bodies. Here we show this state is associated with reduced histamine release, but increased capacity to release bioactive lipids. We describe the overall lipid phenotype of mast cells in this insulin-induced steatotic state and the consequences for critical cellular lipid classes involved in stages of inflammation. We show significant insulin-induced shifts in specific lipid classes, especially arachidonic acid derivatives, MUFA and PUFA, the EPA/DHA ratio, and in cardiolipins, especially those conjugated to certain DHA and EPAs. Functionally, insulin exposure markedly alters the FcεRI-induced release of Series 4 leukotriene LTC4, Series 2 prostaglandin PGD2, Resolvin-D1, Resolvin-D2 and Resolvin-1, reflecting the expanded precursor pools and impact on both the pro-inflammation and pro-resolution bioactive lipids that are released during mast cell activation. Chronic hyperinsulinemia is a feature of obesity and progression to Type 2 Diabetes, these data suggest that mast cell release of key lipid mediators is altered in patients with metabolic syndrome.
Assuntos
Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Insulina/farmacologia , Mastócitos/metabolismo , Animais , Cardiolipinas/metabolismo , Linhagem Celular , Glucose/farmacologia , Mastócitos/efeitos dos fármacos , RatosRESUMO
Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity-based probes as chemical tools for the single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single-cell level.
Assuntos
Corantes Fluorescentes/metabolismo , Staphylococcus aureus/metabolismo , Triazóis/metabolismo , Ureia/metabolismo , Humanos , FenótipoRESUMO
OBJECTIVE: Secretory granules (SG) and lipid bodies (LB) are the primary organelles that mediate functional responses in mast cells. SG contains histamine and matrix-active proteases, while LB are reservoirs of arachidonic acid and its metabolites, precursors for rapid synthesis of eicosanoids such as LTC4. Both of these compartments can be dynamically or ontologically regulated, with metabolic and immunological stimuli altering lipid body content and granule numbers responding to contextual signals from tissue. We previously described that chronic in vitro or in vivo hyperinsulinemia expands the LB compartment with a concomitant loss of SG capacity, suggesting that this ratio is dynamically regulated. The objective of the current study is to determine if chronic insulin exposure initiates a transcriptional program that biases model mast cells towards a lipogenic state with accompanying loss of secretory granule biogenesis. METHODS: We used a basophilic leukemic cell line with mucosal mast cell-like features as a model system. We tested the hypothesis that chronic insulin exposure initiates a transcriptional program that biases these model mast cells towards a lipogenic state with accompanying loss of secretory granule biogenesis. Transcriptional arrays were used to map gene expression patterns. Biochemical, immunocytochemical and mediator release assays were used to evaluate organelle numbers and functional responses. RESULTS: In a mucosal mast cell model, the rat basophilic leukemia line RBL2H3, mast cell granularity and SG numbers are inversely correlated with LB numbers. Chronic insulin exposure appears to modulate gene networks involved in both lipid body biogenesis and secretory granule formation. Western blot analysis confirms upregulation of protein levels for LB proteins, and decreases in proteins that are markers for SG cargo. CONCLUSIONS: The levels of insulin in the extracellular milieu may modify the phenotype of mast cell-like cells in vitro.
RESUMO
IMPORTANCE: Eritoran is a synthetic lipid A antagonist that blocks lipopolysaccharide (LPS) from binding at the cell surface MD2-TLR4 receptor. LPS is a major component of the outer membrane of gram-negative bacteria and is a potent activator of the acute inflammatory response. OBJECTIVE: To determine if eritoran, a TLR4 antagonist, would significantly reduce sepsis-induced mortality. DESIGN, SETTING, AND PARTICIPANTS: We performed a randomized, double-blind, placebo-controlled, multinational phase 3 trial in 197 intensive care units. Patients were enrolled from June 2006 to September 2010 and final follow-up was completed in September 2011. INTERVENTIONS: Patients with severe sepsis (n = 1961) were randomized and treated within 12 hours of onset of first organ dysfunction in a 2:1 ratio with a 6-day course of either eritoran tetrasodium (105 mg total) or placebo, with n = 1304 and n = 657 patients, respectively. MAIN OUTCOME MEASURES: The primary end point was 28-day all-cause mortality. The secondary end points were all-cause mortality at 3, 6, and 12 months after beginning treatment. RESULTS: Baseline characteristics of the 2 study groups were similar. In the modified intent-to-treat analysis (randomized patients who received at least 1 dose) there was no significant difference in the primary end point of 28-day all-cause mortality with 28.1% (366/1304) in the eritoran group vs 26.9% (177/657) in the placebo group (P = .59; hazard ratio, 1.05; 95% CI, 0.88-1.26; difference in mortality rate, -1.1; 95% CI, -5.3 to 3.1) or in the key secondary end point of 1-year all-cause mortality with 44.1% (290/657) in the eritoran group vs 43.3% (565/1304) in the placebo group, Kaplan-Meier analysis of time to death by 1 year, P = .79 (hazard ratio, 0.98; 0.85-1.13). No significant differences were observed in any of the prespecified subgroups. Adverse events, including secondary infection rates, did not differ between study groups. CONCLUSIONS AND RELEVANCE: Among patients with severe sepsis, the use of eritoran, compared with placebo, did not result in reduced 28-day mortality. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00334828.