RESUMEN

Atherosclerosis is a common cardiovascular disease in which the arteries are thickened due to buildup of plaque. This study aims to identify programmed cell death (PCD)-related biomarkers and explore the crucial regulatory mechanisms of atherosclerosis. Gene expression profiles of atherosclerosis and control groups from GSE20129 and GSE23746 were obtained. Necroptosis was elevated in atherosclerosis. Weighted gene coexpression network analysis (WGCNA) was conducted in GSE23746 and GSE56045 to identify PCD-related modules and to perform enrichment analysis. Two necroptosis-related genes (IRF9 and STAT1) were identified and considered as biomarkers. Enrichment analysis showed that these gene modules were mainly related to immune response regulation. In addition, single-cell RNA sequencing data from GSE159677 were obtained and the characteristic cell types of atherosclerosis were identified. A total of 11 immune cell types were identified through UMAP dimension reduction. Most immune cells were mainly enriched in plaque samples, and STAT1 and IRF9 were primarily expressed in T-cells and macrophages. Moreover, the roles of IRF9 and STAT1 were assessed and found to be significantly upregulated in atherosclerosis, which was associated with increased risk of atherosclerosis. This study provides a molecular feature of atherosclerosis, offering an important basis for further research on its pathological mechanisms and the search for new therapeutic targets.

Asunto(s)

Aterosclerosis , Biomarcadores , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón , Factor de Transcripción STAT1 , Linfocitos T , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Humanos , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Biomarcadores/metabolismo , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Redes Reguladoras de Genes , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Macrófagos/inmunología , Macrófagos/metabolismo , Transcriptoma/genética , Placa Aterosclerótica/genética , Placa Aterosclerótica/patología , Placa Aterosclerótica/inmunología

RESUMEN

BACKGROUND: Previous clinical and basic studies have revealed that ginseng might have cardioprotective properties against anthracycline-induced cardiotoxicity (AIC). However, the underlying mechanism of ginseng action against AIC remains insufficiently understood. The aim of this study was to explore the related targets and pathways of ginseng against AIC using network pharmacology, molecular docking, cellular thermal shift assay (CETSA) and molecular dynamics (MD) simulations. RESULTS: Fourteen drug-disease common targets were identified. Enrichment analysis showed that the AGE-RAGE in diabetic complications, fluid shear stress and atherosclerosis, and TNF signaling pathway were potentially involved in the action of ginseng against AIC. Molecular docking demonstrated that the core components including Kaempferol, beta-Sitosterol, and Fumarine had notable binding activity with the three core targets CCNA2, STAT1, and ICAM1. Furthermore, the stable complex of STAT1 and Kaempferol with favorable affinity was further confirmed by CETSA and MD simulation. CONCLUSIONS: This study suggested that ginseng might exert their protective effects against AIC through the derived effector compounds beta-Sitosterol, Kaempferol and Fumarine by targeting CCNA2, STAT1, and ICAM1, and modulating AGE-RAGE in diabetic complications, fluid shear stress and atherosclerosis, and TNF signaling pathways.

Asunto(s)

Antraciclinas , Cardiotoxicidad , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Farmacología en Red , Panax , Panax/química , Antraciclinas/efectos adversos , Antraciclinas/química , Antraciclinas/toxicidad , Humanos , Sitoesteroles/farmacología , Sitoesteroles/química , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Quempferoles/farmacología , Quempferoles/química , Transducción de Señal/efectos de los fármacos

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Factor de Transcripción STAT1 , Trasplante Homólogo , Humanos , Trasplante de Células Madre Hematopoyéticas/métodos , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Masculino , Femenino

RESUMEN

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

Asunto(s)

Sistemas CRISPR-Cas , Dependovirus , Edición Génica , Técnicas de Inactivación de Genes , Factor de Transcripción STAT1 , Humanos , Dependovirus/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Células HEK293 , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Vectores Genéticos/genética , ARN Guía de Sistemas CRISPR-Cas/genética

RESUMEN

BACKGROUND: Obesity is a global epidemic, and the low-grade chronic inflammation of adipose tissue in obese individuals can lead to insulin resistance and type 2 diabetes. Adipose tissue macrophages (ATMs) are the main source of pro-inflammatory cytokines in adipose tissue, making them an important target for therapy. While branched-chain amino acids (BCAA) have been strongly linked to obesity and type 2 diabetes in humans, the relationship between BCAA catabolism and adipose tissue inflammation is unclear. This study aims to investigate whether disrupted BCAA catabolism influences the function of adipose tissue macrophages and the secretion of pro-inflammatory cytokines in adipose tissue, and to determine the underlying mechanism. This research will help us better understand the role of BCAA catabolism in adipose tissue inflammation, obesity, and type 2 diabetes. METHODS: In vivo, we examined whether the BCAA catabolism in ATMs was altered in high-fat diet-induced obesity mice, and if BCAA supplementation would influence obesity, glucose tolerance, insulin sensitivity, adipose tissue inflammation and ATMs polarization in mice. In vitro, we isolated ATMs from standard chow and high BCAA-fed group mice, using RNA-sequencing to investigate the potential molecular pathway regulated by BCAA accumulation. Finally, we performed targeted gene silence experiment and used immunoblotting assays to verify our findings. RESULTS: We found that BCAA catabolic enzymes in ATMs were influenced by high-fat diet induced obesity mice, which caused the accumulation of both BCAA and its downstream BCKA. BCAA supplementation will cause obesity and insulin resistance compared to standard chow (STC) group. And high BCAA diet will induce pro-inflammatory cytokines including Interlukin-1beta (IL-1ß), Tumor Necrosis Factor alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) secretion in adipose tissue as well as promoting ATMs M1 polarization (pro-inflammatory phenotype). Transcriptomic analysis revealed that a high BCAA diet would activate IFNGR1/JAK1/STAT1 pathway, and IFNGR1 specific silence can abolish the effect of BCAA supplementation-induced inflammation and ATMs M1 polarization. CONCLUSIONS: The obesity mice model reveals the catabolism of BCAA was disrupted which will cause the accumulation of BCAA, and high-level BCAA will promote ATMs M1 polarization and increase the pro-inflammatory cytokines in adipose tissue which will cause the insulin resistance in further. Therefore, reducing the circulating level of BCAA can be a therapeutic strategy in obesity and insulin resistance patients.

Asunto(s)

Aminoácidos de Cadena Ramificada , Resistencia a la Insulina , Macrófagos , Obesidad , Factor de Transcripción STAT1 , Transducción de Señal , Animales , Aminoácidos de Cadena Ramificada/metabolismo , Aminoácidos de Cadena Ramificada/administración & dosificación , Macrófagos/metabolismo , Ratones , Masculino , Obesidad/metabolismo , Obesidad/etiología , Factor de Transcripción STAT1/metabolismo , Janus Quinasa 1/metabolismo , Dieta Alta en Grasa/efectos adversos , Tejido Adiposo/metabolismo , Citocinas/metabolismo , Suplementos Dietéticos , Inflamación/metabolismo , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND AND AIMS: Endosialin, also known as tumor endothelial marker1 or CD248, is a transmembrane glycoprotein that is mainly expressed in cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC). Our previous study has found that endosialin-positive CAFs could recruit and induce the M2 polarization of macrophages in HCC. However, whether they may regulate other types of immune cells to promoting HCC progression is not known. APPROACH AND RESULTS: The growth of both subcutaneous and orthotopic HCC tumors was significantly inhibited in endosialin knockout (ENKO) mice. Single-cell sequencing and flow cytometry analysis showed that tumor tissues from ENKO mice had increased CD8+ T cell infiltration. Mixed HCC tumor with Hepa1-6 cells and endosialin knockdown fibroblasts also showed inhibited growth and increased CD8+ T cell infiltration. Data from in vitro co-culture assay, chemokine array and antibody blocking assay, RNA-seq and validation experiments showed that endosialin inhibits the phosphorylation and nuclear translocation of STAT1 in CAFs. This inhibition leads to a decrease in CXCL9/10 expression and secretion, resulting in the suppression of CD8+ T cell infiltration. High level of endosialin protein expression was correlated with low CD8+ T infiltration in the tumor tissue of HCC patients. The combination therapy of endosialin antibody and PD-1 antibody showed synergistic antitumor effect compared with either antibody used individually. CONCLUSIONS: Endosialin could inhibit CD8+ T cell infiltration by inhibiting the expression and secretion of CXCL9/10 in CAFs, thus promote HCC progression. Combination therapy with endosialin antibody could increase the antitumor effect of PD-1 antibody in HCC, which may overcome the resistance to PD-1 blockade.

Asunto(s)

Linfocitos T CD8-positivos , Fibroblastos Asociados al Cáncer , Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Animales , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Antígenos CD/metabolismo , Progresión de la Enfermedad , Línea Celular Tumoral , Quimiocina CXCL9/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones Noqueados , Microambiente Tumoral , Factor de Transcripción STAT1/metabolismo , Quimiocina CXCL10/metabolismo , Masculino , Antígenos de Neoplasias , Proteínas de Neoplasias

RESUMEN

Systemic lupus erythematosus is an autoimmune disease characterized by excessive inflammation and production of pathogenic Abs. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mouse models. In this study, sex- and age-matched wild-type (WT) and HDAC6-/- mice on the C57BL/6 background were administered 0.5 ml of pristane or PBS i.p. at 8-12 wk of age and were euthanized 10 d later. At sacrifice, body weight and spleen weight were measured, sera were collected, and splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT and HDAC6-/- mice. Pristane administration promoted the population of CD11b+Ly6C++ inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils was significantly decreased in HDAC6-/- mice compared with the WT mice. Flow cytometry results showed that the number of CD69+ T and B cells was increased in HDAC6-/- mice. Pristane administration also induced the IFN signature genes as determined by RT-qPCR. Furthermore, IFN signature genes were not affected in HDAC6-/- mice compared with the WT mice. In vitro studies in J774A.1 cells revealed that the selective HDAC6 inhibitor (ACY-738) increased acetylation of NF-κB while increasing Stat1 phosphorylation, which resulted in inducible NO synthase production in LPS/IFN-γ-stimulated cells. Taken together, these results demonstrate that although HDAC6 inhibition may inhibit some inflammatory pathways, others remain unaffected.

Asunto(s)

Histona Desacetilasa 6 , Inflamación , Ratones Endogámicos C57BL , Ratones Noqueados , Terpenos , Animales , Histona Desacetilasa 6/antagonistas & inhibidores , Histona Desacetilasa 6/genética , Histona Desacetilasa 6/metabolismo , Terpenos/farmacología , Ratones , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Bazo/inmunología , Bazo/metabolismo , Bazo/citología , Monocitos/metabolismo , Monocitos/inmunología , Monocitos/efectos de los fármacos , Femenino , Modelos Animales de Enfermedad , Neutrófilos/inmunología , Neutrófilos/metabolismo , Factor de Transcripción STAT1/metabolismo , Masculino , Linfocitos B/inmunología , Linfocitos B/metabolismo

RESUMEN

BACKGROUND: Myocardial ischemia/reperfusion (I/R) injury is a common pathological process in clinical practice. Developing effective therapeutic strategies to reduce or prevent this injury is crucial. The article aimed to investigate the role and mechanism of mesencephalic astrocyte-derived neurotrophic factor (MANF) and its key subdomains in modulating myocardial I/R-induced cardiomyocyte apoptosis. METHODS: MANF stable knockout cell line and MANF mutant overexpression plasmids were constructed. The effects of MANF and mutants on apoptosis and endoplasmic reticulum (ER) stress related proteins were evaluated in hypoxia/reoxygenation-induced HL-1 cardiomyocytes by western blot, immunofluorescence, Tunel and flow cytometry. Echocardiography, ELISA, TTC and Masson were used to observe the effects of recombinant MANF protein (rMANF) on cardiac function in myocardial I/R mice. RESULTS: This study observed increased expression of MANF in both myocardial infarction patients and I/R mice. MANF overexpression in cardiomyocytes decreased ER stress-induced apoptosis, while MANF knockout exacerbated it. rMANF improved cardiac function in I/R mice by reducing injury and inflammation. This study specifically demonstrates that mutations in the α-helix of MANF were more effective in reducing ER stress and cardiomyocyte apoptosis. Mechanistically, MANF and the α-helix mutant attenuated I/R injury by inhibiting the JAK1/STAT1/NF-κB signaling pathway in addition to reducing ER stress-induced apoptosis. CONCLUSION: These findings highlight MANF and its subdomains as critical regulators of myocardial I/R injury, offering promising therapeutic targets with significant clinical implications for I/R-related diseases.

Asunto(s)

Apoptosis , Daño por Reperfusión Miocárdica , Miocitos Cardíacos , Factores de Crecimiento Nervioso , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Línea Celular , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico , Janus Quinasa 1/metabolismo , Janus Quinasa 1/genética , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/genética , Miocitos Cardíacos/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/genética , FN-kappa B/metabolismo , Factor de Transcripción STAT1/metabolismo

RESUMEN

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNß as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

Asunto(s)

Enterovirus Humano B , Miocarditis , Pancreatitis , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Replicación Viral , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Miocarditis/virología , Miocarditis/metabolismo , Miocarditis/patología , Miocarditis/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Animales , Pancreatitis/metabolismo , Pancreatitis/virología , Pancreatitis/patología , Pancreatitis/genética , Enterovirus Humano B/fisiología , Ratones , Humanos , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/patología , Infecciones por Coxsackievirus/genética , Núcleo Celular/metabolismo , Masculino , Transporte Activo de Núcleo Celular , Regulación de la Expresión Génica , Enfermedad Aguda , Línea Celular , Transducción de Señal

RESUMEN

BACKGROUND: Immune checkpoint inhibitors (ICIs) are rapidly evolving in the management of bladder cancer (BLCA). Nevertheless, effective biomarkers for predicting immunotherapeutic outcomes in BLCA are still insufficient. Ferroptosis, a form of immunogenic cell death, has been found to enhance patient sensitivity to ICIs. However, the underlying mechanisms of ferroptosis in promoting immunotherapy efficacy in BLCA remain obscure. METHODS: Our analysis of The Cancer Genome Atlas (TCGA) mRNA data using single sample Gene Set Enrichment Analysis (ssGSEA) revealed two immunologically distinct subtypes. Based on these subtypes and various other public cohorts, we identified Apolipoprotein L6 (APOL6) as a biomarker predicting the efficacy of ICIs and explored its immunological correlation and predictive value for treatment. Furthermore, the role of APOL6 in promoting ferroptosis and its mechanism in regulating this process were experimentally validated. RESULTS: The results indicate that APOL6 has significant immunological relevance and is indicative of immunologically hot tumors in BLCA and many other cancers. APOL6, interacting with acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), mediates immunotherapy efficacy by ferroptosis. Additionally, APOL6 is regulated by signal transducer and activator of transcription 1 (STAT1). CONCLUSIONS: To conclude, our findings indicate APOL6 has potential as a predictive biomarker for immunotherapy treatment success estimation and reveal the STAT1/APOL6/GPX4 axis as a critical regulatory mechanism in BLCA.

Asunto(s)

Biomarcadores de Tumor , Ferroptosis , Inmunoterapia , Neoplasias de la Vejiga Urinaria , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/terapia , Ferroptosis/genética , Humanos , Inmunoterapia/métodos , Biomarcadores de Tumor/genética , Apolipoproteínas/genética , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Animales , Pronóstico , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Ratones

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with an urgent unmet clinical need for new therapies. Using a combination of in vitro assays and in vivo preclinical models we demonstrate that therapeutic inhibition of the IGF signalling axis promotes the accumulation of CD8+ cytotoxic T cells within the tumour microenvironment of PDAC tumours. Mechanistically, we show that IGF blockade promotes macrophage and fibroblast production of the chemokines CXCL9 and CXCL10 to facilitate CD8+ T cell recruitment and trafficking towards the PDAC tumour. Exploring this pathway further, we show that IGF inhibition leads to increased STAT1 transcriptional activity, correlating with a downregulation of the AKT/STAT3 signalling axis, in turn promoting Cxcl9 and Cxcl10 gene transcription. Using patient derived tumour explants, we also demonstrate that our findings translate into the human setting. PDAC tumours are frequently described as "immunologically cold", therefore bolstering CD8+ T cell recruitment to PDAC tumours through IGF inhibition may serve to improve the efficacy of immune checkpoint inhibitors which rely on the presence of CD8+ T cells in tumours.

Asunto(s)

Carcinoma Ductal Pancreático , Quimiocina CXCL10 , Quimiocina CXCL9 , Macrófagos , Neoplasias Pancreáticas , Microambiente Tumoral , Quimiocina CXCL9/metabolismo , Humanos , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Animales , Microambiente Tumoral/inmunología , Quimiocina CXCL10/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Ratones , Somatomedinas/metabolismo , Línea Celular Tumoral , Linfocitos T Citotóxicos/inmunología , Factor de Transcripción STAT1/metabolismo , Linfocitos T CD8-positivos/inmunología , Transducción de Señal , Fibroblastos/metabolismo , Fibroblastos/inmunología , Péptidos Similares a la Insulina

RESUMEN

BACKGROUND: Drug resistance to doxorubicin (DOX) significantly limits its therapeutic efficacy in breast cancer (BC) patients. Saikosaponin D (SSD), a triterpene saponin derived from the traditional herb Radix Bupleuri, has shown promise as a chemotherapeutic sensitizer in preclinical studies due to its notable antitumor activity. However, the role and mechanism of SSD in DOX-resistant BC cells remain largely unexplored. PURPOSE: This study aimed to investigate the chemosensitizing effect of SSD on DOX-resistant BC and the underlying molecular mechanisms both in vitro and in vivo. METHODS: In vitro assays, including cell viability, clone formation, three-dimensional tumor spheroid growth, and apoptosis analysis, were conducted to evaluate the synergistic effect of SSD and DOX on resistant BC cells. Reactive oxygen species (ROS), GSH/GSSG, NADPH/NADP+, and NADH/NAD+ detections were employed to assess the impact of SSD on cellular redox homeostasis. Western blotting, cell cycle distribution assay, and DOX uptake assay were performed to further elucidate the possible antineoplastic mechanism of SSD. Finally, a subcutaneous MCF7/DOX cell xenografted model in nude mice was established to identify the in vivo anticarcinogenic effect of SSD combined with DOX. RESULTS: SSD significantly inhibited cell viability, proliferation, and clone formation, enhancing DOX's anticancer efficacy in vitro and in vivo. Mechanistically, SSD reduced STAT1, NQO1, and PGC-1α protein levels, leading to cellular redox imbalance, excessive ROS generation, and depletion of GSH, NADPH, and NADH. SSD induced DNA damage by disrupting redox homeostasis, resulting in G0/G1 phase cell cycle arrest. Additionally, SSD increased DOX accumulation in BC cells via inhibiting P-gp protein expression and efflux activity. CONCLUSION: We demonstrated for the first time that SSD enhances the sensitivity of chemoresistant BC cells to DOX by disrupting cellular redox homeostasis through inactivation of the STAT1/NQO1/PGC-1α signaling pathway. This study provides evidence for SSD as an adjuvant agent in drug-resistant BC treatment.

Asunto(s)

Neoplasias de la Mama , Doxorrubicina , Resistencia a Antineoplásicos , Ratones Desnudos , NAD(P)H Deshidrogenasa (Quinona) , Ácido Oleanólico , Oxidación-Reducción , Especies Reactivas de Oxígeno , Saponinas , Doxorrubicina/farmacología , Saponinas/farmacología , Ácido Oleanólico/farmacología , Ácido Oleanólico/análogos & derivados , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Femenino , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratones , Sinergismo Farmacológico , Células MCF-7 , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de Xenoinjerto , Factor de Transcripción STAT1/metabolismo , Supervivencia Celular/efectos de los fármacos , Antineoplásicos Fitogénicos/farmacología

RESUMEN

BACKGROUND: By identifying molecular biological markers linked to cuproptosis in diabetic retinopathy (DR), new pathobiological pathways and more accessible diagnostic markers can be developed. METHODS: The datasets related to DR were acquired from the Gene Expression Omnibus database, while genes associated with cuproptosis were sourced from previously published compilations. Consensus clustering was conducted to delineate distinct DR subclasses. Feature genes were identified utilizing weighted correlation network analysis (WGCNA). Additionally, two machine-learning algorithms were employed to refine the selection of feature genes. Finally, we conducted preliminary validation experiments to ascertain the involvement of cuproptosis in DR development and the transcriptional regulation of critical genes using both the streptozotocin-induced diabetic mouse model and the high glucose-induced BV2 model. RESULTS: In the STZ-induced diabetic mouse retinas, a decrease in the expression of cuproptosis signature proteins (FDX1, DLAT, and NDUFS8) suggested the occurrence of cuproptosis in DR. Subsequently, the expression of eight cuproptosis differential genes was validated through the STZ-induced diabetes and oxygen-induced retinopathy (OIR) models, with the key gene SLC31A1 showing upregulation in both models and dataset species. Further analyses, including weighted gene co-expression network analysis, GSVA, and immune infiltration analysis, indicated a close correlation between cuproptosis and microglia function. Additionally, validation in an in vitro model of microglia indicated the occurrence of cuproptosis in microglia under high glucose conditions, alongside abnormal expression of STAT1 with SLC31A1. CONCLUSION: Our findings suggest that STAT1/SLC31A1 may pave the way for a deeper comprehension of the mechanistic basis of DR and offer potential therapeutic avenues.

Asunto(s)

Diabetes Mellitus Experimental , Retinopatía Diabética , Factor de Transcripción STAT1 , Animales , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Ratones , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Masculino , Retina/metabolismo , Retina/patología , Ratones Endogámicos C57BL , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos

RESUMEN

Objective To explore the effects of Myxovirus resistance protein A (MxA) on the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway in HepG2 cells. Methods HepG2 cells were transfected with the pcDNA3.1-Flag-MxA construct, and subsequent localization and expression of the MxA protein were detected through immunofluorescence cytochemistry. The presence of MxA protein was further confirmed by using Western blot analysis. Following transfection with MxA small interfering RNA (si-MxA) and subsequent treatment with alpha interferon (IFN-α), real-time fluorescent quantitative PCR was employed to measure the mRNA levels of myxovirus resistance protein A (MxA), protein kinase R (PKR), and oligoadenylate synthase (OAS). Western blot analysis was used to detect the protein expression of MxA, PKR, OAS, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1), STAT2, phosphorylated STAT2 (p-STAT2) and interferon regulatory factor 9 (IRF9). Additionally, pcDNA3.1-Flag-MxA and pISRE-TA-luc were co-transfected into HepG2 and HepG2.2.15 cells, respectively, to assess the activity of the interferon-stimulated response element (ISRE) by using a luciferase activity assay. Results MxA protein was expressed in both the cytoplasm and nucleus of HepG2 cells, with higher expression levels in the cytoplasm than in the nucleus. Knocking down MxA expression in HepG2 cells did not affect the expression of STAT1, p-STAT1, STAT2, p-STAT2, and IRF9 proteins induced by IFN-α, but significantly reduced the expression of antiviral proteins PKR and OAS. Overexpression of MxA in HepG2 cells enhanced ISRE activity and increased the expression of PKR and OAS proteins, but this effect was inhibited in HepG2.2.15 cells. Conclusion MxA induces the expression of antiviral proteins by enhancing the activity of the JAK/STAT signaling pathway ISRE.

Asunto(s)

2',5'-Oligoadenilato Sintetasa , Proteínas de Resistencia a Mixovirus , Factor de Transcripción STAT1 , eIF-2 Quinasa , Humanos , Células Hep G2 , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Interferón-alfa/farmacología , Interferón-alfa/genética , Interferón-alfa/metabolismo , Elementos de Respuesta/genética , Transducción de Señal , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Interferones/genética , Interferones/metabolismo , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Regulación de la Expresión Génica

RESUMEN

Changes in mechanosensitive ion channels following radiation have seldom been linked to therapeutic sensitivity or specific factors involved in antitumor immunity. Here, in this study, we found that the mechanical force sensor, Piezo2, was significantly upregulated in tumor cells after radiation, and Piezo2 knockout in tumor cells enhanced tumor growth suppression by radiotherapy. Specifically, loss of Piezo2 in tumor cells induced their IL-15 expression via unleashing JAK2/STAT1/IRF-1 axis after radiation. This increase in IL-15 activates IL-15Rα on tumor-infiltrating CD8+ T cells, thereby leading to their augmented effector and stem cell-like properties, along with reduced terminal exhausted feature. Importantly, Piezo2 expression was negatively correlated with CD8 infiltration, as well as with radiosensitivity of patients with rectum adenocarcinoma receiving radiotherapy treatment. Together, our findings reveal that tumor cell-intrinsic Piezo2 induces radioresistance by dampening the IRF-1/IL-15 axis, thus leading to impaired CD8+ T cell-dependent antitumor responses, providing insights into the further development of combination strategies to treat radioresistant cancers.

Asunto(s)

Linfocitos T CD8-positivos , Interleucina-15 , Canales Iónicos , Tolerancia a Radiación , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Animales , Humanos , Canales Iónicos/metabolismo , Canales Iónicos/genética , Tolerancia a Radiación/genética , Ratones , Interleucina-15/metabolismo , Interleucina-15/genética , Línea Celular Tumoral , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 1 Regulador del Interferón/genética , Ratones Endogámicos C57BL , Femenino , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Masculino , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Transducción de Señal

RESUMEN

DNA damage has been implicated in the stimulation of the type 1 interferon (T1IFN) response. Here, we show that downregulation of the DNA repair protein, polynucleotide kinase/phosphatase (PNKP), in a variety of cell lines causes robust phosphorylation of STAT1, upregulation of interferon-stimulated genes and persistent accumulation of cytosolic DNA, all of which are indicators for the activation of the T1IFN response. Furthermore, this did not require damage induction by ionizing radiation. Instead, our data revealed that production of reactive oxygen species (ROS) synergises with PNKP loss to potentiate the T1IFN response, and that loss of PNKP significantly compromises mitochondrial DNA (mtDNA) integrity. Depletion of mtDNA or treatment of PNKP-depleted cells with ROS scavengers abrogated the T1IFN response, implicating mtDNA as a significant source of the cytosolic DNA required to potentiate the T1IFN response. The STING signalling pathway is responsible for the observed increase in the pro-inflammatory gene signature in PNKP-depleted cells. While the response was dependent on ZBP1, cGAS only contributed to the response in some cell lines. Our data have implications for cancer therapy, since PNKP inhibitors would have the potential to stimulate the immune response, and also to the neurological disorders associated with PNKP mutation.

Asunto(s)

Enzimas Reparadoras del ADN , ADN Mitocondrial , Interferón Tipo I , Fosfotransferasas (Aceptor de Grupo Alcohol) , Radiación Ionizante , Especies Reactivas de Oxígeno , Humanos , Interferón Tipo I/metabolismo , Interferón Tipo I/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Reparación del ADN , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Daño del ADN , Línea Celular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Fosforilación , Citosol/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética

RESUMEN

Due to the urgent need to create appropriate treatment techniques, which are currently unavailable, LPS-induced sepsis has become a serious concern on a global scale. The primary active component in the pathophysiology of inflammatory diseases such as sepsis is the Gram-negative bacterial lipopolysaccharide (LPS). LPS interacts with cell surface TLR4 in macrophages, causing the formation of reactive oxygen species (ROS), TNF-α, IL-1ß and oxidative stress. It also significantly activates the MAPKs and NF-κB pathway. Excessive production of pro-inflammatory cytokines is one of the primary characteristic features in the onset and progression of inflammation. Cytokines mainly signal through the JAK/STAT pathway. We hypothesize that blocking of TLR4 along with TNFR1 might be beneficial in suppressing the effects of STAT1/STAT3 due to the stimulation of SOCS3 proteins. Prior to the LPS challenge, the macrophages were treated with antibodies against TLR4 and TNFR1 either individually or in combination. On analysis of the macrophage populations by flowcytometry, it was seen that receptor blockade facilitated the phenotypic shift of the M1 macrophages towards M2 resulting in lowered oxidative stress. Blocking of TLR4/TNFR1 upregulated the SOCS3 and mTOR expressions that enabled the transition of inflammatory M1 macrophages towards the anti-inflammatory M2 phenotype, which might be crucial in curbing the inflammatory responses. Also the reduction in the production of inflammatory cytokines such as IL-6, IL-1ß due to the reduction in the activation of the STAT1 and STAT3 molecules was observed in our combination treatment group. All these results indicated that neutralization of both TLR4 and TNFR1 might provide new insights in establishing an alternative therapeutic strategy for LPS-sepsis.

Asunto(s)

Lipopolisacáridos , Macrófagos , Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Proteína 3 Supresora de la Señalización de Citocinas , Receptor Toll-Like 4 , Receptor Toll-Like 4/metabolismo , Animales , Macrófagos/inmunología , Macrófagos/metabolismo , Factor de Transcripción STAT1/metabolismo , Ratones , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Citocinas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Activación de Macrófagos/efectos de los fármacos

RESUMEN

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity, research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast, cancer cell-intrinsic PD-1 functional regulation is not well understood. Here, we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-ß triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling, increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer, and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors.

Asunto(s)

Inhibidores de Puntos de Control Inmunológico , Interferón Tipo I , Melanoma , Receptor de Muerte Celular Programada 1 , Receptor de Interferón alfa y beta , Transducción de Señal , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melanoma/genética , Melanoma/metabolismo , Humanos , Receptor de Interferón alfa y beta/metabolismo , Receptor de Interferón alfa y beta/genética , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Ratones , Interferón Tipo I/metabolismo , Factor de Transcripción STAT1/metabolismo , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Interferón beta/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Quinasas Janus/metabolismo , Ratones Endogámicos C57BL , Interferón-alfa/farmacología , Interferón-alfa/metabolismo , Femenino

RESUMEN

Severe aplastic anemia (SAA) is a life-threatening bone marrow failure syndrome whose development can be triggered by environmental, autoimmune, and/or genetic factors. The latter comprises germ line pathogenic variants in genes that bring about habitually predisposing syndromes as well as immune deficiencies that do so only occasionally. One of these disorders is the autosomal dominant form of chronic mucocutaneous candidiasis (CMC), which is defined by germ line STAT1 gain-of-function (GOF) pathogenic variants. The resultant overexpression and constitutive activation of STAT1 dysregulate the Janus kinase/signal transducer and activator of transcription 1 (STAT) signaling pathway, which normally organizes the development and proper interaction of different components of the immunologic and hematopoietic system. Although SAA is an extremely rare complication in this disorder, it gained a more widespread interest when it became clear that the underlying causative pathomechanism may, in a similar fashion, also be instrumental in at least some of the idiopathic SAA cases. Based on these premises, we present herein what is the historically most likely first cord blood-transplanted SAA case in a CMC family with a documented STAT1 GOF pathogenic variant. In addition, we recapitulate the characteristics of the six CMC SAA cases that have been reported so far and discuss the significance of STAT1 GOF pathogenic variants and other STAT1 signaling derangements in the context of these specific types of bone marrow failure syndromes. Because a constitutively activated STAT1 signaling, be it driven by STAT1 GOF germ line pathogenic variants or any other pathogenic variant-independent events, is apparently important for initiating and maintaining the SAA disease process, we propose to acknowledge that SAA is one of the definite disease manifestations in STAT1-mutated CMC cases. For the same reason, we deem it necessary to also incorporate molecular and functional analyses of STAT1 into the diagnostic work-up of SAA cases.

Asunto(s)

Anemia Aplásica , Candidiasis Mucocutánea Crónica , Factor de Transcripción STAT1 , Adulto , Femenino , Humanos , Masculino , Anemia Aplásica/genética , Candidiasis Mucocutánea Crónica/genética , Trasplante de Células Madre de Sangre del Cordón Umbilical , Linaje , Estudios Retrospectivos , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo

RESUMEN

OBJECTIVE: To explore the role and molecular mechanism of cancer-associated fibroblasts (CAFs) in the tumor microenvironment of gastric cancer (GC). METHODS: The expression of CAFs in GC patients was first assessed for abundance, and survival analysis was performed. Subsequently, The Cancer Genome Atlas (TCGA) data were used for differential analysis, survival analysis, and EPIC analysis, while single-cell data (GSE183904) were downloaded for differential analysis of CAFs. Clinical data pooling, univariate and multivariate Cox analysis, and immunofluorescence were carried out on clinical GC tissue samples to explore RCN3 expression within patient CAFs. Western blot and quantitative polymerase chain reaction (qPCR) were used to detect the expression of RCN3. The relationship between RCN3, PCSK6, and STAT1 was explored by chromatin immunoprecipitation (CHIP) experiments, and the effects of the genes on macrophage polarization were detected by detecting biomarkers of biological M1/M2. RESULTS: CAFs in GC were found to be significantly higher compared to the normal group. Revealing the results of TCGA differential analysis, it was observed that GC exhibited a substantial upregulation in the expression levels of RCN3. The clinical statistics indicate a positive correlation between an elevated level of RCN3 expression and the T-stage classification of tumor size. In addition, RCN3 was found to have a significant impact on the overall survival of patients with gastric cancer, acting as an independent prognostic indicator. Analysis of single-cell data showed high expression of PCSK6 in macrophages, and immunofluorescence staining of samples from GC patients showed increased expression of PCSK6 on the cell membranes of macrophages in GC tissues. The subsequent cellular experiments confirmed RCN3 protein can regulate the expression of PCSK6, and PCSK6 regulates macrophage polarization through STAT1. CONCLUSIONS: CAFs regulate macrophage polarization through the RCN3/PCSK6/STAT1 pathway in GC.

Asunto(s)

Fibroblastos Asociados al Cáncer , Macrófagos , Neoplasias Gástricas , Microambiente Tumoral , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Microambiente Tumoral/genética , Macrófagos/metabolismo , Femenino , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Regulación Neoplásica de la Expresión Génica , Masculino , Transducción de Señal , Persona de Mediana Edad , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Activación de Macrófagos/genética