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Background: It is unclear whether patients with metabolic dysfunction-associated steatotic liver disease (MASLD) are allowed variable low levels of alcohol. This study aimed to evaluate the effect of mild-moderate alcohol consumption on the biochemical and histological characteristics of patients with MASLD. Methods: Alcohol consumption was assessed in 713 patients with steatotic liver disease (SLD) who underwent liver biopsy. Non-drinking, mild-moderate drinking, and excessive drinking were defined as 0 g/day, 1-<20 g/day, and >20 g/day for women and 0 g/day, 1-<30 g/day, and >30 g/day for men, respectively. Liver biopsies were scored according to the NASH CRN system. Results: A total of 713 participants (median age 39.0 years and 77.1% male) with biopsy-proven SLD were enrolled, including 239 nondrinkers, 269 mild-moderate drinkers and 205 excessive drinkers. Excessive drinking was associated with increased risks for lobular inflammation and liver fibrosis compared to nondrinkers and mild-moderate drinkers. Compared with non-drinkers, mild-moderate drinkers had significantly lower odds for steatosis (OR = 0.60, 95% CI = 0.38-0.93, p = 0.025), hepatocellular ballooning (OR = 0.52, 95% CI = 0.29-0.91, p = 0.020) and fibrosis (OR = 0.50, 95% CI = 0.31-0.81, p = 0.005). However, in non-excessive drinkers with type 2 diabetes mellitus (T2DM), there was no association between mild-moderate alcohol consumption and liver fibrosis (OR = 0.562, 95% CI = 0.207-1.530, p = 0.257). Conclusions: Mild-moderate alcohol consumption might be protective against liver fibrosis in MASLD patients, which is modified by the presence of T2DM. However, further longitudinal studies are needed to determine the effect of ongoing alcohol consumption on disease severity.
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OBJECTIVE: To explore the clinical effect of percutaneous pedicle screw anchored vertebral augmentation(PPSAVA) in the treatment of asymptomatic Kümmell disease without neurological symptoms. METHODS: The clinical data of 20 patients with Kümmell disease without neurological symptoms treated with PPSAVA in our hospital from January 2019 to December 2021 were analyzed retrospectively, including 5 males and 15 females, aged 56 to 88 (74.95±9.93) years old. and the course of disease was 7 to 60 days with an average of (21.35±14.46) days. All patients were treated with PPSAVA. The time of operation, the amount of bone cement injected and the leakage of bone cement were recorded. The visual analogue scale(VAS), Oswestry disability index(ODI), vertebral body angle(VBA), anterior edge height and midline height of vertebral body were compared among the before operation, 3 days after operation and during the final follow-up. The loosening and displacement of bone cement were observed during the final follow-up. RESULTS: All the 20 patients completed the operation successfully. The operation time was 30 to 56 min with an average of (41.15±7.65) min, and the amount of bone cement injection was 6.0 to 12.0 ml with an average of (9.30±1.49) ml. Bone cement leakage occurred in 6 cases and there were no obvious clinical symptoms. The follow-up time was 6 to 12 months with an average of (8.43±2.82) months. The VBA, anterior edge height and midline height of of injured vertebral body were significantly improved 3 days after operation and the final follow-up(P<0.05), and the VBA, anterior edge height and midline height of of injured vertebral body were lost in different degrees at the final follow-up (P<0.05). The VAS and ODI at 3 days after operation and at the final follow-up were significantly lower than those at preoperatively(P<0.05), but the VAS score and ODI at the final follow-up were not significantly different from those at 3 d after operation(P>0.05). At the last follow-up, no patients showed loosening or displacement of bone cement. CONCLUSION: PPSAVA is highly effective in treating Kümmell disease without neurological symptoms, improving patients' pain and functional impairment, and reducing the risk of cement loosening and displacement postoperatively.
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Tornillos Pediculares , Humanos , Femenino , Masculino , Anciano , Persona de Mediana Edad , Anciano de 80 o más Años , Estudios Retrospectivos , Fracturas de la Columna Vertebral/cirugía , Cementos para HuesosRESUMEN
BACKGROUND: Our study investigated the role of FAM53B in regulating macrophage M2 polarization and its potential mechanisms in promoting pancreatic ductal adenocarcinoma (PDAC) metastasis. AIM: To further investigate the role of FAM53B in regulating macrophage M2 polarization and its potential mechanism in promoting PDAC metastasis. Our goal is to determine how FAM53B affects macrophage M2 polarization and to define its underlying mechanism in PDAC metastasis. METHODS: Cell culture and various experiments, including protein analysis, immunohistochemistry, and animal model experiments, were conducted. We compared FAM53B expression between PDAC tissues and healthy tissues and assessed the correlation of FAM53B expression with clinical features. Our study analyzed the role of FAM53B in macrophage M2 polarization in vitro by examining the expression of relevant markers. Finally, we used a murine model to study the role of FAM53B in PDAC metastasis and analyzed the potential underlying mechanisms. RESULTS: Our research showed that there was a significant increase in FAM53B levels in PDAC tissues, which was linked to adverse tumor features. Experimental findings indicated that FAM53B can enhance macrophage M2 polarization, leading to increased anti-inflammatory factor release. The results from the mouse model further supported the role of FAM53B in PDAC metastasis, as blocking FAM53B prevented tumor cell invasion and metastasis. CONCLUSION: FAM53B promotes PDAC metastasis by regulating macrophage M2 polarization. This discovery could lead to the development of new strategies for treating PDAC. For example, interfering with the FAM53B signaling pathway may prevent cancer spread. Our research findings also provide important information for expanding our understanding of PDAC pathogenesis.
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BACKGROUND: There is growing recognition of natural history, complications, and outcomes of patients who develop non-acetaminophen (APAP) drug-induced acute liver failure (ALF). To clarify high-risk factors and develop a nomogram model to predict transplant-free survival (TFS) in patients with non-APAP drug-induced ALF. METHODS: Patients with non-APAP drug-induced ALF from 5 participating centers were retrospectively analyzed. The primary endpoint was 21-day TFS. Total sample size was 482 patients. RESULTS: Regarding causative agents, the most common implicated drugs were herbal and dietary supplements (HDS) (57.0%). The hepatocellular type (R ≥ 5) was the main liver injury pattern (69.0%). International normalized ratio, hepatic encephalopathy grades, the use of vasopressor, N-acetylcysteine, or artificial liver support system were associated with TFS and incorporated to construct a nomogram model (drug-induced acute liver failure-5, DIALF-5). The AUROC of DIALF-5 for 7-day, 21-day, 60-day, and 90-day TFS in the internal cohort were 0.886, 0.915, 0.920, and 0.912, respectively. Moreover, the AUROC of DIALF-5 for 21-day TFS had the highest AUROC, which was significantly higher than 0.725 of MELD and 0.519 of KCC (p < 0.05), numerically higher than 0.905 of ALFSG-PI but without statistical difference (p > 0.05). These results were successfully validated in the external cohort (147 patients). CONCLUSIONS: Based on easily identifiable clinical data, the novel DIALF-5 model was developed to predict transplant-free survival in non-APAP drug-induced ALF, which was superior to KCC, MELD and had a similar prediction performance to ALFSG-PI but is more convenient, which can directly calculate TFS at multiple time points.
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Fallo Hepático Agudo , Humanos , Estudios Retrospectivos , Pronóstico , Fallo Hepático Agudo/etiología , Nomogramas , Factores de RiesgoRESUMEN
Constitutively active mutations in the Gαi2 and GαoA subunits of heterotrimeric G proteins have been found in various human cancers, including breast cancer, but their precise roles in tumor formation, progression, and metastasis remain poorly understood. This study focused on GαoAR243H and Gαi2R179C mutants in breast cancer. These mutants alone were insufficient to initiate mammary tumor formation in mice. However, when introduced into transgenic mouse models of breast cancer induced by Neu expression or PTEN loss, the Gαi2R179C mutant notably enhanced spontaneous lung metastasis, without affecting primary tumor initiation and growth. Ectopic expression of the GαoAR243H and Gαi2R179C mutants in tumor cells promoted cell migration in vitro and dissemination into multiple organs in vivo by activating the c-Src signaling pathway. These mutants activate c-Src through direct interaction, involving specific residues in the switch domains II of Gαi subunits, which only partially overlap with those involved in inhibiting adenylyl cyclases. This study uncovers a critical role of Gαi/o signaling in accelerating breast cancer metastasis through the c-Src pathway. These findings hold clinical significance as they may pave the way for personalized therapies targeting c-Src to inhibit breast cancer metastasis in patients with active Gαi/o mutations or elevated Gαi/o signaling.
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Neoplasias de la Mama , Transducción de Señal , Ratones , Humanos , Animales , Femenino , Transducción de Señal/fisiología , Ratones Transgénicos , Neoplasias de la Mama/metabolismo , Metástasis de la NeoplasiaRESUMEN
Constitutively active mutations in the Gαi2 and GαoA subunits of heterotrimeric G proteins have been identified in several human cancers including breast cancer, but their functional significance in tumorigenesis and metastasis has not been well characterized. In this study, we show that expression of the constitutively active GαoAR243H and Gαi2R179C mutants alone was insufficient to induce mammary tumor formation in mice. However, in transgenic mouse models of breast cancer induced by Neu expression or PTEN loss, we found that the Gαi2R179C mutant enhanced spontaneous lung metastasis while having no effect on primary tumor initiation and growth. Additionally, we observed that ectopic expression of the GαoAR243H and Gαi2R179C mutants in tumor cells promote cell migration in vitro as well as dissemination into multiple organs in vivo by activating c-Src signaling. Thus, our study uncovers a critical function of Gαi/o signaling in accelerating breast cancer metastasis via the c-Src pathway. This work is clinically significant, as it can potentially pave the way to personalized therapies for patients who present with active Gαi/o mutations or elevated Gαi/o signaling by targeting c-Src to inhibit breast cancer metastasis.
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Hepatocellular carcinoma (HCC) is a highly aggressive malignant disease, and numerous studies have shown that certain functional long noncoding RNAs (lncRNAs) are implicated in the progression of several cancers. The purpose of the research was to determine, using a database, bioinformatics, and statistical analysis, whether or not lncRNA PRR7-AS1 (PRR7-AS1) was related to HCC. TCGA datasets were used to conduct research on the PRR7-AS1 expression pattern in HCC. In order to evaluate the efficacy of GIHCG as a prognostic tool, both survival and Cox regression analyses were carried out. Furthermore, an investigation of the connection between the expression of PRR7-AS1 and immune infiltration in HCC was carried out. In this study, we identified 125 lncRNAs that were significantly dysregulated in HCC and were associated with long-term survival. Among the above 125 lncRNAs, our attention focused on PRR7-AS1. We found that PRR7-AS1 expressions were distinctly overexpressed in HCC samples compared with nontumor samples. ROC assays revealed that PRR7-AS1 effectively differentiated HCC specimens from normal tissues with an AUC of 0.875 (95% CI: 0.840 to 0.911). Moreover, the high PRR7-AS1 expression was associated with advanced clinical stage and poor prognosis of HCC patients. Importantly, the multivariate Cox proportional hazards model suggested that up-expression of PRR7-AS1 was an independent prognostic marker indicating shorter overall survival and disease-specific survival for HCC patients. Finally, we found that PRR7-AS1 expression was associated with the expression of NK CD56bright cells, Th2 cells, TFH, macrophages, Th1 cells, aDC, T helper cells, cytotoxic cells, DC, Tgd, neutrophils, and Th17 cells. Overall, the results of our study show that PRR7-AS1 was a biomarker that could be utilized to predict the prognosis of HCC patients and was linked to the infiltration of immune cells in HCC.
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INTRODUCTION AND IMPORTANCE: Primary hepatic neuroendocrine tumors (PHNETs) are extremely rare, and the clinical symptoms, test results, and imaging characteristics are nonspecific in most patients; thus, it is difficult to differentiate from other liver masses before surgery. Histopathology and immunohistochemistry are the main basis for the diagnosis. PHNETs and colon tumors co-occur in a patient and are non-homologous, as reported in the English-language literature for the first time. CASE PRESENTATION: We present a case of a 60-year-old woman with right hepatic lobe mass accidentally discovered on abdominal ultrasonography during a routine examination. Preoperative liver contrast-enhanced computed tomography suggested hepatocellular carcinoma; then, surgery were performed. Pathological results revealed a Grade 2 neuroendocrine tumor of the liver. In search of the primary tumor, upper and lower endoscopy of the GI tract was performed and revealed a mass in the ascending colon. Ascending colon cancer was considered; then, laparoscopic right hemicolectomy was performed. Pathological results suggested tubular villous adenoma of the ascending colon. The final diagnosis was not colon cancer with liver metastases but was PHNETs with colon adenoma. CLINICAL DISCUSSION: PHNETs are rare cancers that are difficult to diagnose, requiring not only differentiation from other liver masses but also exclusion of metastases from extrahepatic sources. The pathological results play an important in making an accurate diagnosis. CONCLUSION: Pathology, postoperative follow-up, and comprehensive imaging examinations are powerful tools in the diagnosis of PHNETs. Currently, surgery is the best treatment to achieve a potential cure and prolong the patient's survival.
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Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that are resistant to anti-tumor therapies, making them a likely origin of tumor relapse after treatment. In many cancers including breast cancer, CSC function is regulated by G protein-coupled receptors (GPCRs), making GPCR signaling an attractive target for new therapies designed to eradicate CSCs. Yet, CSCs overexpress multiple GPCRs that are redundant in maintaining CSC function, so it is unclear how to target all the various GPCRs to prevent relapse. Here, in a model of HER2+ breast cancer (i.e., transgenic MMTV-Neu mice), we were able to block the tumorsphere- and tumor-forming capability of CSCs by targeting GPCRs coupled to Gi/o proteins (Gi/o-GPCRs). Similarly, in HER2+ breast cancer cells, blocking signaling downstream of Gi/o-GPCRs in the PI3K/AKT and Src pathways also enhanced HER2-targeted elimination of CSCs. In a proof-of-concept study, when CSCs were selectively ablated (via a suicide gene construct), loss of CSCs from HER2+ breast cancer cell populations mimicked the effect of targeting Gi/o-GPCR signaling, suppressing their capacity for tumor initiation and progression and enhancing HER2-targeted therapy. Thus, targeting Gi/o-GPCR signaling in HER2+ breast cancer is a promising approach for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor reemergence.
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Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4-labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.
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Estratos Germinativos/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Seudópodos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas de Pez Cebra/metabolismo , Actinas/metabolismo , Animales , Embrión no Mamífero/metabolismo , Endodermo/metabolismo , Estratos Germinativos/embriología , Glicosilfosfatidilinositoles/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Mesodermo/embriología , Mesodermo/metabolismo , Transporte de Proteínas , Pez CebraRESUMEN
GPCRs are highly desirable drug targets for human disease. Although GPCR dysfunction drives development and progression of many tumors, including breast cancer (BC), targeting individual GPCRs has limited efficacy as a cancer therapy because numerous GPCRs are activated. Here, we sought a new way of blocking GPCR activation in HER2+ BC by targeting a subgroup of GPCRs that couple to Gi/o proteins (Gi/o-GPCRs). In mammary epithelial cells of transgenic mouse models, and BC cell lines, HER2 hyperactivation altered GPCR expression, particularly, Gi/o-GPCR expression. Gi/o-GPCR stimulation transactivated EGFR and HER2 and activated the PI3K/AKT and Src pathways. If we uncoupled Gi/o-GPCRs from their cognate Gi/o proteins by pertussis toxin (PTx), then BC cell proliferation and migration was inhibited in vitro and HER2-driven tumor formation and metastasis were suppressed in vivo. Moreover, targeting Gi/o-GPCR signaling via PTx, PI3K, or Src inhibitors enhanced HER2-targeted therapy. These results indicate that, in BC cells, HER2 hyperactivation drives aberrant Gi/o-GPCR signaling and Gi/o-GPCR signals converge on the PI3K/AKT and Src signaling pathways to promote cancer progression and resistance to HER2-targeted therapy. Our findings point to a way to pharmacologically deactivate GPCR signaling to block tumor growth and enhance therapeutic efficacy.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Glándulas Mamarias Animales/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antineoplásicos Inmunológicos/farmacología , Benzodioxoles/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Epitelio/metabolismo , Receptores ErbB/metabolismo , Femenino , Humanos , Indazoles/farmacología , Lapatinib/farmacología , Ratones Transgénicos , Metástasis de la Neoplasia , Toxina del Pertussis , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Trastuzumab/farmacología , Regulación hacia ArribaRESUMEN
BACKGROUND AND AIMS: About 20% of patients receiving nucleos(t)ide analogues treatment experienced low-level viraemia (LLV), which is associated with progression of liver fibrosis and high risk of hepatocellular carcinoma. We aimed to evaluate the effectiveness and safety of switching from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) in ETV-treated patients with LLV. METHODS: In this prospective study, ETV-treated patients with LLV, presented to our hospital from December 2018 to October 2019, were enrolled. Switching to TAF or continuing ETV was given. The primary effectiveness endpoint was complete virological response (CVR) at 24 weeks, and the safety endpoint was the first occurrence of any clinical adverse event during the treatment. RESULTS: Totally, 211 patients were recruited and propensity score matching (PSM) generated 75 patients in either TAF or ETV group. After PSM, baseline characteristics were balanced in two groups. After 24-week treatment, the CVR and ALT normalization in TAF group were 62.7% and 47.6%, which were higher than 9.3% and 10.5% in ETV group (OR 16.4, 95% CI 6.6-40.0, P < .001) respectively. Subgroup analysis showed that switching to TAF achieved favours CVR regardless of the status of sex, age, CHB family history, HBV DNA, HBeAg and cirrhosis, whereas alcohol consumption and diabetes mellitus might compromise the CVR of switching to TAF. Both therapies were well tolerated and had satisfying renal safety. CONCLUSIONS: For ETV-treated patients with LLV, switching to TAF is safe enough and superior compared with continuing ETV monotherapy regarding both virological and biochemical benefits.
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Hepatitis B Crónica , Adenina/análogos & derivados , Alanina , Antivirales/efectos adversos , Guanina/análogos & derivados , Virus de la Hepatitis B , Hepatitis B Crónica/tratamiento farmacológico , Humanos , Estudios Prospectivos , Tenofovir/análogos & derivados , Resultado del Tratamiento , Viremia/tratamiento farmacológicoRESUMEN
SOX2 and OCT4 are key regulators of embryonic stem cell pluripotency. They are overexpressed in prostate cancers and have been associated with cancer stem cell (CSC) properties. However, reliable tools for detecting and targeting SOX2/OCT4-overexpressing cells are lacking, limiting our understanding of their roles in prostate cancer initiation, progression, and therapeutic resistance. Here, we show that a fluorescent reporter called SORE6 can identify SOX2/OCT4-overexpressing prostate cancer cells. Among tumor cells, the SORE6 reporter identified a small fraction with CSC hallmarks: rapid self-renewal, the capability to form tumors and metastasize, and resistance to chemotherapies. Transcriptome and biochemical analyses identified PI3K/AKT signaling as critical for maintaining the SORE6+ population. Moreover, a SORE6-driven herpes simplex virus thymidine kinase (TK) expression construct could selectively ablate SORE6+ cells in tumors, blocking tumor initiation and progression, and sensitizing tumors to chemotherapy. This study demonstrates a key role of SOX2/OCT4-associated prostate cancer stem cells in tumor development and therapeutic resistance, and identifies the SORE6 reporter system as a useful tool for characterizing CSCs functions in a native tumor microenvironment.
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Obesity is associated with elevated intracellular nitric oxide (NO) production, which promotes nitrosative stress in metabolic tissues such as liver and skeletal muscle, contributing to insulin resistance. The onset of obesity-associated insulin resistance is due, in part, to the compromise of hepatic autophagy, a process that leads to lysosomal degradation of cellular components. However, it is not known how NO bioactivity might impact autophagy in obesity. Here, we establish that S-nitrosoglutathione reductase (GSNOR), a major protein denitrosylase, provides a key regulatory link between inflammation and autophagy, which is disrupted in obesity and diabetes. We demonstrate that obesity promotes S-nitrosylation of lysosomal proteins in the liver, thereby impairing lysosomal enzyme activities. Moreover, in mice and humans, obesity and diabetes are accompanied by decreases in GSNOR activity, engendering nitrosative stress. In mice with a GSNOR deletion, diet-induced obesity increases lysosomal nitrosative stress and impairs autophagy in the liver, leading to hepatic insulin resistance. Conversely, liver-specific overexpression of GSNOR in obese mice markedly enhances lysosomal function and autophagy and, remarkably, improves insulin action and glucose homeostasis. Furthermore, overexpression of S-nitrosylation-resistant variants of lysosomal enzymes enhances autophagy, and pharmacologically and genetically enhancing autophagy improves hepatic insulin sensitivity in GSNOR-deficient hepatocytes. Taken together, our data indicate that obesity-induced protein S-nitrosylation is a key mechanism compromising the hepatic autophagy, contributing to hepatic insulin resistance.
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Alcohol Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/metabolismo , Autofagia , Diabetes Mellitus/metabolismo , Hepatocitos/metabolismo , Resistencia a la Insulina , Obesidad/fisiopatología , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/genética , Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/genética , Animales , Células Cultivadas , Cisteína/metabolismo , Diabetes Mellitus/enzimología , Diabetes Mellitus/patología , Dieta Alta en Grasa/efectos adversos , Regulación Enzimológica de la Expresión Génica , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hepatocitos/enzimología , Hepatocitos/patología , Humanos , Lisosomas/enzimología , Lisosomas/metabolismo , Lisosomas/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Estrés Nitrosativo , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de FusiónRESUMEN
Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gß1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gßγ signaling, neutrophils failed to migrate to wound sites. Although both the Gß1 and Gß4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gß1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gß1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gß1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gß1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gß isoform in chemotaxis in vivo.
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Quimiotaxis de Leucocito/fisiología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Neutrófilos/fisiología , Cicatrización de Heridas/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Subunidades beta de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades beta de la Proteína de Unión al GTP/genética , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/genética , Morfolinos/genética , Transducción de Señal , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Aberrant activation of G protein-coupled receptors (GPCRs) is implicated in prostate cancer progression, but targeting them has been challenging because multiple GPCRs are involved in cancer progression. In this study, we tested the effect of blocking signaling via a hub through which multiple GPCRs converge - the G-protein Gßγ subunits. Inhibiting Gßγ signaling in several castration-resistant prostate cancer cell lines (i.e. PC3, DU145 and 22Rv1), impaired cell growth and migration in vitro, and halted tumor growth and metastasis in nude mice. The blockade of Gßγ signaling also diminished prostate cancer stem cell-like activities, by reducing tumorsphere formation in vitro and tumor formation in a limiting dilution assay in nude mice. Furthermore, Gßγ blockade enhanced the sensitivity of prostate cancer cells to paclitaxel treatment, both in vitro and in vivo. Together, our results identify a novel function of Gßγ in regulating prostate cancer stem-cell-like activities, and demonstrate that targeting Gßγ signaling is an effective approach in blocking prostate cancer progression and augmenting response to chemotherapy.
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Antineoplásicos/uso terapéutico , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células Madre Neoplásicas/fisiología , Paclitaxel/uso terapéutico , Neoplasias de la Próstata/metabolismo , Animales , Carcinogénesis , Procesos de Crecimiento Celular , Línea Celular Tumoral , Movimiento Celular , Resistencia a Antineoplásicos , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/tratamiento farmacológico , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The phosphatidylinositol 3-kinase (PI3K)/AKT pathway transmits signals downstream of receptor tyrosine kinases and G protein-coupled receptors (GPCRs), and is one of the most dysregulated pathways in breast cancer. PI3Ks and AKTs consist of multiple isoforms that play distinct and even opposite roles in breast cancer cell growth and metastasis. However, it remains unknown how the activities of various PI3K and AKT isoforms are coordinated during breast cancer progression. Previously, we showed WDR26 is a novel WD40 protein that binds Gßγ and promotes Gßγ signaling. Here, we demonstrate that WDR26 is overexpressed in highly malignant breast tumor cell lines and human breast cancer samples, and that WDR26 overexpression correlates with shortened survival of breast cancer patients. In highly malignant cell lines (MDA-MB231, DU4475 and BT549), downregulation of WDR26 expression selectively alleviated GPCR- but not EGF receptor-stimulated PI3K/AKT signaling and tumor cell growth, migration and invasion. In contrast, in a less malignant cell line (MCF7), WDR26 overexpression had the opposite effect. Additional studies indicate that downstream of GPCR stimulation, WDR26 serves as a scaffold that fosters assembly of a specific signaling complex consisting of Gßγ, PI3Kß and AKT2. In an orthotopic xenograft mouse model of breast cancer, disrupting formation of this complex, by overexpressing WDR26 mutants in MDA-MB231 cells, abrogated PI3K/AKT activation and tumor cell growth and metastasis. Together, our results identify a novel mechanism regulating GPCR-dependent activation of the PI3K/AKT signaling axis in breast tumor cells, and pinpoint WDR26 as a potential therapeutic target for breast cancer.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Femenino , Xenoinjertos , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Invasividad Neoplásica , Transducción de Señal , Regulación hacia ArribaRESUMEN
Rearrangement of the actin cytoskeleton is controlled by RhoGTPases which are activated by RhoGEFs. We identified homozygosity for Arg204Trp mutation in the Rho guanidine exchange factor (RhoGEF) PLEKHG2 gene in five patients with profound mental retardation, dystonia, postnatal microcephaly, and distinct neuroimaging pattern. The activity of the mutant PLEKHG2 was significantly decreased, both in basal state and when Gßγ- or lysophosphatidic acid (LPA)-stimulated. SDF1a-stimulated actin polymerization was significantly impaired in patient cells, and this abnormality was duplicated in control cells when PLEKHG2 expression was downregulated. These results underscore the role of PLEKHG2 in actin polymerization and delineate the clinical and radiological findings in PLEKHG2 deficiency.
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Actinas/metabolismo , Distonía/genética , Factores de Intercambio de Guanina Nucleótido/genética , Microcefalia/genética , Árabes , Consanguinidad , Distonía/complicaciones , Familia , Femenino , Células HEK293 , Humanos , Masculino , Microcefalia/complicaciones , Medio Oriente , Mutación Missense , Linaje , Multimerización de Proteína/genéticaRESUMEN
Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , FN-kappa B/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Ligando de CD40/fisiología , Caspasa 8/metabolismo , Dominio Catalítico , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Resistencia a Antineoplásicos , Células HEK293 , Enfermedad de Hodgkin/metabolismo , Humanos , Células Jurkat , Ratones Noqueados , Datos de Secuencia Molecular , Proteolisis , UbiquitinaciónRESUMEN
Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.