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OBJECTIVE: To investigate the role of bacterial- mediated plasminogen (PLG) activation in the pathogenesis of anastomotic leak (AL) and its mitigation by tranexamic acid (TXA). BACKGROUND: AL is the most feared complication of colorectal resections. The pathobiology of AL in the setting of a technically optimal procedure involves excessive submucosal collagen degradation by resident microbes. We hypothesized that activation of the host PLG system by pathogens is a central and targetable pathway in AL. METHODS: We employed kinetic analysis of binding and activation of human PLG by microbes known to cause AL, and collagen degradation assays to test the impact of PLG on bacterial collagenolysis. Further, we measured the ability of the antifibrinolytic drug TXA to inhibit this process. Finally, using mouse models of pathogen-induced AL, we locally applied TXA via enema and measured its ability to prevent a clinically relevant AL. RESULTS: PLG is deposited rapidly and specifically at the site of colorectal anastomoses. TXA inhibited PLG activation and downstream collagenolysis by pathogens known to have a causal role in AL. TXA enema reduced collagenolytic bacteria counts and PLG deposition at anastomotic sites. Postoperative PLG inhibition with TXA enema prevented clinically and pathologically apparent pathogen-mediated AL in mice. CONCLUSIONS: Bacterial activation of host PLG is central to collagenolysis and pathogen-mediated AL. TXA inhibits this process both in vitro and in vivo. TXA enema represents a promising method to prevent AL in high-risk sites such as the colorectal anastomoses.
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Fístula Anastomótica/microbiologia , Fístula Anastomótica/prevenção & controle , Colo/cirurgia , Plasminogênio/metabolismo , Ácido Tranexâmico/administração & dosagem , Animais , Colágeno/efeitos dos fármacos , Modelos Animais de Doenças , Enema , Enterococcus faecalis , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pseudomonas aeruginosaRESUMO
BACKGROUND & AIMS: The Western diet, which is high in fat, is a modifiable risk factor for colorectal recurrence after curative resection. We investigated the mechanisms by which the Western diet promotes tumor recurrence, including changes in the microbiome, in mice that underwent colorectal resection. METHODS: BALB/c male mice were fed either standard chow diet or Western-type diet (characterized by high fat, no fiber, and decreased minerals and vitamins) for 4 weeks; some mice were given antibiotics or ABA-PEG20k-Pi20 (Pi-PEG), which inhibits collagenase production by bacteria, but not bacterial growth, in drinking water. Colorectal resections and anastomoses were then performed. The first day after surgery, mice were given enemas containing a collagenolytic rodent-derived strain of Enterococcus faecalis (strain E2), and on the second day they were given mouse colon carcinoma cells (CT26). Twenty-one days later, distal colons were removed, and colon contents (feces, distal colon, and tumor) were collected. Colon tissues were analyzed by histology for the presence of collagenolytic colonies and by 16S ribosomal RNA sequencing, which determined the anatomic distribution of E faecalis at the site of the anastomosis and within tumors using in situ hybridization. Mouse imaging analyses were used to identify metastases. RESULTS: Colorectal tumors were found in 88% of mice fed the Western diet and given antibiotics, surgery, and E faecalis compared with only 30% of mice fed the standard diet followed by the same procedures. Colon tumor formation correlated with the presence of collagenolytic E faecalis and Proteus mirabilis. Antibiotics eliminated collagenolytic E faecalis and P mirabilis but did not reduce tumor formation. However, antibiotics promoted emergence of Candida parapsilosis, a collagenase-producing microorganism. Administration of a Pi-PEG reduced tumor formation and maintained diversity of the colon microbiome. CONCLUSIONS: We identified a mechanisms by which diet and antibiotic use can promote tumorigenesis by colon cancer cells at the anastomosis after colorectal surgery. Strategies to prevent emergence of these microbe communities or their enzymatic activities might be used to reduce the risk of tumor recurrence in patients undergoing colorectal cancer surgery.
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Colectomia/efeitos adversos , Neoplasias Colorretais/microbiologia , Dieta Ocidental/efeitos adversos , Microbioma Gastrointestinal , Complicações Pós-Operatórias/microbiologia , Protectomia/efeitos adversos , Anastomose Cirúrgica/efeitos adversos , Animais , Antibacterianos/uso terapêutico , Carcinogênese , Colágeno , Enterococcus faecalis/crescimento & desenvolvimento , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos OrgânicosRESUMO
This study demonstrates that independent additive effects of two human pharmaceuticals, the antibiotic trimethoprim and the artificial estrogen 17a-Ethinylestradiol (EE2), inhibit plant litter decomposition by aquatic microorganisms. The constant release of pharmaceuticals, such as these, has the potential to affect aquatic microbial metabolism and alter biogeochemical cycling of carbon and nutrients. Here we advance the Tea Bag Index (TBI) for decomposition by using it in a series of contaminant exposure experiments testing how interactions between trimethoprim and EE2 affect aquatic microbial activity. The TBI is a citizen science tool used to test microbial activity by measuring the differential degradation of green and rooibos tea as proxies for respectively labile and recalcitrant litter decomposition. Exposure to either trimethoprim or EE2 decreased decomposition of green tea, suggesting additive effects upon microbial activity. Exposure to EE2 alone decreased rooibos tea decomposition. Consequently, trimethoprim and EE2 stabilized labile organic matter against microbial degradation and restricted decomposition. We propose that the method outlined could provide a powerful tool for testing the impacts of multiple interacting pollutants upon microbial activity, at a range of scales, across aquatic systems and over ecologically relevant time scales.
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Ecossistema , Preparações Farmacêuticas , Carbono , Humanos , Folhas de Planta , Plantas , CháRESUMO
Despite advances in surgical technique and the expanded use of antibiotics, anastomotic leak remains a dreaded complication leading to increased hospital length of stay, morbidity, mortality, and cost. Data continues to grow addressing the importance of a functional and diverse colonic microbiome to ensure adequate healing. Individual pathogens, such as Enterococcus faecalis and Pseudomonas aeruginosa , have been implicated in the pathogenesis of anastomotic leak. Yet how these pathogens proliferate remains unclear. It is possible that decreased microbial diversity promotes a shift to a pathologic phenotype among the remaining microbiota which may lead to anastomotic breakdown. As the microbiome is highly influenced by diet, antibiotic use, the stress of surgery, and opioid use, these factors may be modifiable at various phases of the surgical process. A large amount of data remains unknown about the composition and behavior of the "normal" gut microbiome as compared with an altered community. Therefore, targeting the gut microbiome as a modifiable factor in anastomotic healing may represent a novel strategy for the prevention of anastomotic leak.
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Perforations, anastomotic leak, and subsequent intra-abdominal sepsis are among the most common and feared complications of invasive interventions in the colon and remaining intestinal tract. During physiological healing, tissue protease activity is finely orchestrated to maintain the strength and integrity of the submucosa collagen layer in the wound. We (Shogan, BD et al. Sci Trans Med 7: 286ra68, 2015.) have previously demonstrated in both mice and humans that the commensal microbe Enterococcus faecalis selectively colonizes wounded colonic tissues and disrupts the healing process by amplifying collagenolytic matrix-metalloprotease activity toward excessive degradation. Here, we demonstrate for the first time, to our knowledge, a novel collagenolytic virulence mechanism by which E. faecalis is able to bind and locally activate the human fibrinolytic protease plasminogen (PLG), a protein present in high concentrations in healing colonic tissue. E. faecalis-mediated PLG activation leads to supraphysiological collagen degradation; in this study, we demonstrate this concept both in vitro and in vivo. This pathoadaptive response can be mitigated with the PLG inhibitor tranexamic acid (TXA) in a fashion that prevents clinically significant complications in validated murine models of both E. faecalis- and Pseudomonas aeruginosa-mediated colonic perforation. TXA has a proven clinical safety record and is Food and Drug Administration approved for topical application in invasive procedures, albeit for the prevention of bleeding rather than infection. As such, the novel pharmacological effect described in this study may be translatable to clinical trials for the prevention of infectious complications in colonic healing.NEW & NOTEWORTHY This paper presents a novel mechanism for virulence in a commensal gut microbe that exploits the human fibrinolytic system and its principle protease, plasminogen. This mechanism is targetable by safe and effective nonantibiotic small molecules for the prevention of infectious complications in the healing gut.
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Colágeno Tipo IV/metabolismo , Colágeno Tipo I/metabolismo , Colo/microbiologia , Enterococcus faecalis/metabolismo , Fibrinólise , Infecções por Bactérias Gram-Positivas/microbiologia , Plasminogênio/metabolismo , Infecção da Ferida Cirúrgica/microbiologia , Cicatrização , Animais , Antibacterianos/farmacologia , Antifibrinolíticos/farmacologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/patogenicidade , Fibrinólise/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/patologia , Infecções por Bactérias Gram-Positivas/prevenção & controle , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos C57BL , Plasminogênio/antagonistas & inibidores , Proteólise , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/prevenção & controle , Infecção da Ferida Cirúrgica/metabolismo , Infecção da Ferida Cirúrgica/patologia , Infecção da Ferida Cirúrgica/prevenção & controle , Ácido Tranexâmico/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Virulência , Cicatrização/efeitos dos fármacosRESUMO
Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior.
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Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Aprendizagem/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Neurônios/patologia , Fosforilação , Fosfosserina/metabolismo , Biossíntese de Proteínas , Serina/genética , Serina/metabolismo , TransgenesRESUMO
Thoracoabdominal aneurysms pose technical challenges for endovascular repair due to involvement of visceral and renal vessels. We report a case series of four patients diagnosed with thoracoabdominal aneurysm who underwent complex endovascular repair with Fenestrated Device and chimney grafts (FEVARCh). FEVARCh is a technically feasible approach for repair of thoracoabdominal aneurysms that involve renal, superior mesenteric, and celiac arteries for patients not appropriate for open surgical repair. Further studies are needed to understand the implications of resultant Type 1a endoleaks and strategies to minimize the displacement of the main body graft with adjunct chimneys.
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Aneurisma da Aorta Abdominal , Aneurisma da Aorta Torácica , Implante de Prótese Vascular , Procedimentos Endovasculares , Aneurisma da Aorta Abdominal/cirurgia , Aneurisma da Aorta Torácica/diagnóstico por imagem , Aneurisma da Aorta Torácica/cirurgia , Prótese Vascular , Implante de Prótese Vascular/efeitos adversos , Procedimentos Endovasculares/efeitos adversos , Humanos , Complicações Pós-Operatórias/cirurgia , Desenho de Prótese , Estudos Retrospectivos , Fatores de Risco , Stents , Resultado do TratamentoRESUMO
Purpose: Large studies have demonstrated improved survival outcomes with thoracic endovascular aortic repair (TEVAR) at two and five years compared to medical therapy; however, early TEVAR for acute type B aortic dissection (TBAD) remains controversial. We aimed to evaluate trends and clinical predictors of hospital readmissions in patients undergoing medical management and TEVAR for acute TBADs. Materials and Methods: The Nationwide Readmissions Database was queried for all 30-day and 90-day index readmissions (30D-IR and 90D-IR, respectively) after a diagnosis of a TBAD from January 2012 to September 2015. Data on readmission diagnosis, patient demographics, and hospital characteristics were collected from readmitted patients and analyzed. Multivariable logistic regression models were used to identify the predictors of readmission after TEVAR or medical medical management of TBAD. Results: We identified 53,117 patients with acute TBAD. Medical management was the initial treatment modality in 46,985 (88.4%) patients, while 6,132 (11.5%) underwent TEVAR. Factors including older patient age, lower household income, severity of comorbidities, initial hospital length of stay, and urgent procedure demonstrated an increased likelihood of experiencing 30D-IR and 90D-IR (P<0.05). The rate of unplanned readmission for patients undergoing medical management remained stable (11.3% vs. 10.0% for 30D-IR; 19.1% vs. 15.5% for 90D-IR). Reasons for unplanned readmission in the TEVAR cohort were largely related to technical complications. There was no significant difference in readmission costs between medical management and TEVAR. Conclusion: Number of unplanned readmissions in the TEVAR arm decreased significantly over time, whereas the number of readmissions for medical management remained stable.
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Much about the role of intestinal microbes at the site of colon cancer development and tumor progression following curative resection remains to be understood. We have recently shown that collagenolytic bacteria such as Enterococcus faecalis predominate within the colon postoperatively, particularly at the site of the colon reconnection (i.e. anastomosis) in the early period of post-surgical recovery. The presence of collagenolytic bacteria at this site correlates with the tumor progression in a mouse model of post-surgical tumor development. In the present study we hypothesized, that collagenolytic bacteria, such as E. faecalis, play an important yet to be discovered role in tumor formation and progression. Therefore the aims of this study were to assess the role of collagenolytic E. faecalis on the migration and invasion of a murine colon cancer cell line. Results demonstrated that both migration and invasion were induced by E. faecalis with collagenolytic activity being required for only invasion. Bidirectional signaling in the E. faecalis-cancer cell interaction was observed by the discovering that the expression of gelE in E. faecalis, the gene required for collagenase production, is expressed in response to exposure to CT26 cells. The mechanism by which migration enhancement via E. faecalis occurs appears to be dependent on its ability to activate pro-uPA, a key element of the urokinase-plasminogen system, a pathway that is well - known to be important in cancer cell invasion and migration. Finally, we demonstrated that collagenase producing microbes preferentially colonize human colon cancer specimens.
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Neoplasias do Colo , Enterococcus faecalis , Animais , Colagenases/metabolismo , Neoplasias do Colo/genética , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Humanos , Camundongos , Fenótipo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
We present the successful endovascular repair of an iatrogenic aortic dissection in a 57-year-old woman with decompensated heart failure. An intra-aortic balloon pump was inserted in the patient via a percutaneous axillary approach for circulatory support. Six days later, she developed symptoms of abdominal pain and lower extremity malperfusion. Computed tomography angiography demonstrated a type B aortic dissection with associated retroperitoneal hematoma secondary to aortic perforation. The patient underwent emergent endovascular aortic repair and intra-aortic balloon pump removal with return of lower extremity perfusion. She recovered well and underwent heart and kidney transplant less than 2 months postoperatively.
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Framing uncertain scenarios to emphasize potential positive or negative elements influences decision making and behavior. The current experiment investigated sex differences in framing effects on risk-taking propensity in a modified version of the Balloon Analogue Risk Task (BART). Male and female undergraduates completed questionnaires on sensation seeking, impulsiveness, and risk and benefit perception prior to viewing one of three framing conditions for the BART: (1) positively-framed instructions emphasizing the ability to earn money if balloons were inflated to large size; (2) negatively framed instructions emphasizing the possibility that money could be lost if balloons were inflated to bursting; and (3) completely framed instructions noting both possible outcomes. Results revealed correlations between BART performance and impulsiveness for both sexes. Compared to positive and complete framing, negatively framed instructions decreased balloon inflation time in women but not men, indicating sex differences in response to treatments designed to alter risk-taking behavior.
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Comportamento Impulsivo/psicologia , Assunção de Riscos , Adulto , Tomada de Decisões , Feminino , Humanos , Masculino , Análise de Regressão , Autorrelato , Fatores Sexuais , Inquéritos e QuestionáriosRESUMO
BACKGROUND: Previous work from our group has suggested a pivotal role for collagenolytic bacteria in the development of anastomotic complications. Tumor necrosis factor antagonists are a mainstay of treatment for patients with inflammatory bowel disease. The reported impact of these agents on key surgical outcomes such as anastomotic leak has been inconsistent. The objective of this study is to assess the impact of infliximab on the anastomotic microbiome in a mouse model of colon resection. DESIGN: BALB/c mice underwent colon resection with primary anastomosis. Mice were randomly assigned to receive either an intraperitoneal dose of saline (control) or 10 mg/kg of infliximab for 8 weeks prior to surgery. On postoperative day 7, the animals were sacrificed. Anastomotic tissues were analyzed by histology with TUNNEL staining as a marker of epithelial apoptosis. In order to assess compositional and functional changes of the local microbiome, anastomotic tissues were further analyzed by 16S rRNA V4 region sequencing and for the presence of collagenolytic strains that may impair anastomotic healing. The main outcome measures were microbiome community structure and the presence of collagenolytic bacteria. RESULTS: Infliximab-treated mice demonstrated an increase in epithelial apoptosis, consistent with the expected drug effect. Although infliximab modified the perianastomotic microbiome, no increase in the presence of collagenolytic bacteria was observed. CONCLUSIONS: Infliximab did not promote the emergence of collagenolytic bacteria or demonstrably impair anastomotic healing in a mouse model of colon resection and anastomosis.
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Fístula Anastomótica , Neoplasias Colorretais , Anastomose Cirúrgica , Fístula Anastomótica/etiologia , Animais , Bactérias , Colo/cirurgia , Humanos , Infliximab , Camundongos , Camundongos Endogâmicos BALB C , RNA Ribossômico 16SRESUMO
INTRODUCTION: Endovascular approach to abdominal aortic aneurysm has increased in frequency over the last decade and is preferred by many practitioners and patients alike. However, its role in "complex" aneurysms with challenging anatomy, short neck length, or borderline landing zone for grafts is less well understood. Additionally, the endovascular role in complex urgent or emergent repairs has not been adequately studied. Several techniques have been developed in order to address these concerns and further the endovascular approach. This article seeks to describe incidents in which an endovascular approach may be challenging, describe the most popular techniques now available for approaching these cases, and present the best literature available for considering these techniques. EVIDENCE ACQUISITION: Medline searches were performed with the key words "complex endovascular aneurysm repair," "fenestrated endovascular aneurysm repair," "chimney grafts" and "snorkel grafts" to gain access to available data. EVIDENCE SYNTHESIS: Articles were reviewed for results and technical considerations. CONCLUSIONS: By employing increasingly complex endovascular techniques, complex pararenal aneurysms can be successfully treated in an endovascular fashion.
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Aneurisma da Aorta Abdominal/cirurgia , Implante de Prótese Vascular , Procedimentos Endovasculares , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/mortalidade , Aneurisma da Aorta Abdominal/fisiopatologia , Prótese Vascular , Implante de Prótese Vascular/efeitos adversos , Implante de Prótese Vascular/instrumentação , Implante de Prótese Vascular/mortalidade , Procedimentos Endovasculares/efeitos adversos , Procedimentos Endovasculares/instrumentação , Procedimentos Endovasculares/mortalidade , Humanos , Complicações Pós-Operatórias/etiologia , Desenho de Prótese , Stents , Resultado do TratamentoRESUMO
p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKTS473/T308 and S6K1T389 phosphorylation but decreased S6S235/236 and IRS-1S1101 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on ob/ob and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in ob/ob and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKTS473/T308 phosphorylation in the fat and muscle of ob/ob mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity in vitro, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.
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Glicemia/efeitos dos fármacos , Hiperglicemia/prevenção & controle , Leflunomida/farmacologia , Células 3T3-L1 , Compostos de Anilina/farmacologia , Animais , Glicemia/metabolismo , Células Cultivadas , Crotonatos , Glucose/metabolismo , Teste de Tolerância a Glucose , Hidroxibutiratos/farmacologia , Hiperglicemia/sangue , Leflunomida/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Nitrilas , Obesidade/sangue , Obesidade/complicações , Obesidade/patologia , Ratos , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , ToluidinasRESUMO
mTOR activation suppresses autophagy by phosphorylating ULK1 at S757 and suppressing its enzymatic activity. Here we report that feedback activation of mTOR in the PI-3 kinase pathway by two p70 S6 kinase (S6K1) inhibitors (PF-4708671 and A77 1726, the active metabolite of an immunosuppressive drug leflunomide) or by S6K1 knockdown did not suppress but rather induced autophagy. Suppression of S6K1 activity led to the phosphorylation and activation of AMPK, which then phosphorylated ULK1 at S555. While mTOR feedback activation led to increased phosphorylation of ULK1 at S757, this modification did not the disrupt ULK1-AMPK interaction nor dampen ULK1 S555 phosphorylation and the induction of autophagy. In addition, inhibition of S6K1 activity led to JNK activation, which also contributed to autophagy. 5Z-7-oxozeaenol, a specific inhibitor of TAK1, or TAK1 siRNA blocked A77 1726-induced activation of AMPK and JNK, and LC3 lipidation. Taken together, our study establishes S6K1 as a key player in the PI-3 kinase pathway to suppress autophagy through inhibiting AMPK and JNK in a TAK1-dependent manner.
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Compostos de Anilina/farmacologia , Autofagia/efeitos dos fármacos , Hidroxibutiratos/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Crotonatos , Técnicas de Silenciamento de Genes , Humanos , MAP Quinase Quinase Quinases/genética , Modelos Biológicos , Mutação , Nitrilas , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Transdução de Sinais , ToluidinasRESUMO
The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.
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Movimento Celular/genética , Proteínas Hedgehog/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Caderinas/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cromonas/farmacologia , Ativação Enzimática , Transição Epitelial-Mesenquimal/genética , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Morfolinas/farmacologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail/efeitos dos fármacos , Fatores de Transcrição da Família Snail/genética , Alcaloides de Veratrum/farmacologia , Vimentina/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
CONTEXT: Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood. OBJECTIVE: The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms. DESIGN: The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay. RESULTS: Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells. CONCLUSIONS: The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.
Assuntos
Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/fisiologia , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/metabolismo , Aldeído Desidrogenase/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Fatores de Transcrição da Família Snail , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor-mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 µM, respectively. Exogenous uridine partially blocked A77 1726-induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726-induced cell cycle arrest in the S phase.
Assuntos
Compostos de Anilina/farmacologia , Ciclo Celular/efeitos dos fármacos , Hidroxibutiratos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Butadienos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cromonas/farmacologia , Crotonatos , Retroalimentação Fisiológica , Técnicas de Silenciamento de Genes , Humanos , Imidazóis/farmacologia , Isoxazóis/farmacologia , Leflunomida , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfolinas/farmacologia , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Pirazinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , ToluidinasAssuntos
Neoplasias Colorretais , Protocolos de Quimioterapia Combinada Antineoplásica , Quimioterapia Adjuvante , Tomada de Decisão Clínica , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Humanos , Terapia Neoadjuvante , Guias de Prática Clínica como Assunto , Ensaios Clínicos Controlados Aleatórios como Assunto , Estados UnidosRESUMO
Shewanellae are microbial models for environmental stress response; however, the sequential expression of mechanisms in response to stress is poorly understood. Here we experimentally determine the response mechanisms of Shewanella amazonensis SB2B during sodium chloride stress using a novel liquid chromatography and accurate mass-time tag mass spectrometry time-course proteomics approach. The response of SB2B involves an orchestrated sequence of events comprising increased signal transduction associated with motility and restricted growth. Following a metabolic shift to branched chain amino acid degradation, motility and cellular replication proteins return to pre-perturbed levels. Although sodium chloride stress is associated with a change in the membrane fatty acid composition in other organisms, this is not the case for SB2B as fatty acid degradation pathways are not expressed and no change in the fatty acid profile is observed. These findings suggest that shifts in membrane composition may be an indirect physiological response to high NaCl stress.