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Mycobacteria are causative agents of tuberculosis (TB), which is a global health concern. Drug-resistant TB strains are rapidly emerging, thereby necessitating the urgent development of new drugs. Two-component signal transduction systems (TCSs) are signaling pathways involved in the regulation of various bacterial behaviors and responses to environmental stimuli. Applying specific inhibitors of TCSs can disrupt bacterial signaling, growth, and virulence, and can help combat drug-resistant TB. We conducted a comprehensive pharmacophore-based inhibitor screening and biochemical and biophysical examinations to identify, characterize, and validate potential inhibitors targeting the response regulators PhoP and MtrA of mycobacteria. The constructed pharmacophore model Phar-PR-n4 identified effective inhibitors of formation of the PhoP-DNA complex: ST132 (IC50 = 29 ± 1.6 µM) and ST166 (IC50 = 18 ± 1.3 µM). ST166 (KD = 18.4 ± 4.3 µM) and ST132 (KD = 14.5 ± 0.1 µM) strongly targeted PhoP in a slow-on, slow-off manner. The inhibitory potency and binding affinity of ST166 and ST132 for MtrAC were comparable to those of PhoP. Structural analyses and molecular dynamics simulations revealed that ST166 and ST132 mainly interact with the α8-helix and C-terminal ß-hairpin of PhoP, with functionally essential residue hotspots for structure-based inhibitor optimization. Moreover, ST166 has in vitro antibacterial activity against Macrobacterium marinum. Thus, ST166, with its characteristic 1,2,5,6-tetrathiocane and terminal sulphonic groups, has excellent potential as a candidate for the development of novel antimicrobial agents to combat pathogenic mycobacteria.
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BACKGROUND: Interstitial lung disease (ILD) treatment is a critical unmet need. Selenium is an essential trace element for human life and an antioxidant that activates glutathione, but the gap between its necessity and its toxicity is small and requires special attention. Whether selenium can be used in the treatment of ILD remains unclear. METHODS: We investigated the prophylactic and therapeutic effects of selenite, a selenium derivative, in ILD using a murine model of bleomycin-induced idiopathic pulmonary fibrosis (IPF). We further elucidated the underlying mechanism using in vitro cell models and examined their relevance in human tissue specimens. The therapeutic effect of selenite in bleomycin-administered mice was assessed by respiratory function and histochemical changes. Selenite-induced apoptosis and reactive oxygen species (ROS) production in murine lung fibroblasts were measured. RESULTS: Selenite, administered 1 day (inflammation phase) or 8 days (fibrotic phase) after bleomycin, prevented and treated deterioration of lung function and pulmonary fibrosis in mice. Mechanistically, selenite inhibited the proliferation and induced apoptosis of murine lung fibroblasts after bleomycin treatment both in vitro and in vivo. In addition, selenite upregulated glutathione reductase (GR) and thioredoxin reductase (TrxR) in murine lung fibroblasts, but not in lung epithelial cells, upon bleomycin treatment. GR and TrxR inhibition eliminates the therapeutic effects of selenite. Furthermore, we found that GR and TrxR were upregulated in the human lung fibroblasts of IPF patient samples. CONCLUSIONS: Selenite induces ROS production and apoptosis in murine lung fibroblasts through GR and TrxR upregulation, thereby providing a therapeutic effect in bleomycin-induced IPF.
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Apoptosis , Bleomicina , Fibroblastos , Especies Reactivas de Oxígeno , Ácido Selenioso , Bleomicina/efectos adversos , Animales , Ratones , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Ácido Selenioso/farmacología , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/metabolismo , Modelos Animales de Enfermedad , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Masculino , Proliferación Celular/efectos de los fármacosRESUMEN
Cancer immunotherapy harnesses the immune system to combat tumors and has emerged as a major cancer treatment modality. The PD-1/PD-L1 immune checkpoint modulates interactions between tumor cells and T cells and has been extensively targeted in cancer immunotherapy. However, the monoclonal antibodies known to target this immune checkpoint have considerable side effects, and novel PD-1/PD-L1 inhibitors are therefore required. Herein, a peptide inhibitor to disrupt PD-1/PD-L1 interactions was designed through structure-driven phage display engineering coupled to computational modification and optimization. BetaPb, a novel peptide library constructed by using the known structure of PD-1/PD-L, was used to develop inhibitors against the immune checkpoint, and specific peptides with high affinity toward PD-1 were screened through enzyme-linked immunosorbent assays, homogeneous time-resolved fluorescence, and biolayer interferometry. A potential inhibitor, B8, was preliminarily screened through biopanning. The binding affinity of B8 toward PD-1 was confirmed through computation-aided optimization. Assessment of B8 variants (B8.1, B8.2, B8.3, B8.4, and B8.5) demonstrated their attenuation of PD-1/PD-L1 interactions. B8.4 exhibited the strongest attenuation efficiency at a half-maximal effective concentration of 0.1 µM and the strongest binding affinity to PD-1 (equilibrium dissociation constant = 0.1 µM). B8.4 outperformed the known PD-1/PD-L1 interaction inhibitor PL120131 in disrupting PD-1/PD-L1 interactions, revealing that B8.4 has remarkable potential for modification to yield an antitumor agent. This study provides valuable information for the future development of peptide-based drugs, therapeutics, and immunotherapies for cancer.
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Bacteriófagos , Neoplasias , Humanos , Inhibidores de Puntos de Control Inmunológico , Receptor de Muerte Celular Programada 1/química , Antígeno B7-H1/química , Péptidos/farmacología , Péptidos/química , Bacteriófagos/metabolismoRESUMEN
This study aimed to evaluate the protein expression of glutathione peroxidase 4 (GPX4) in resected non-small cell lung cancer (NSCLC). The clinical relevance and prognostic significance of GPX4 expression were analyzed. We reviewed patients with resected NSCLCs at Taipei Veterans General Hospital between September 2002 and January 2018. Available paraffin-embedded specimens were retrieved for immunohistochemistry (IHC) staining to detect GPX4 expression. The cutoff value for defining GPX4 positivity was determined according to the percentage of tumor stained in the microscopic field. The correlation between immune expression, clinicopathologic data, overall survival (OS), and disease-free survival (DFS) were analyzed. A total of 265 NSCLC specimens were retrieved for IHC staining. GPX4 expression positive was in 192 (72.5%) according to a cutoff value of 5%. GPX4 was a significant prognostic factor for OS and DFS on multivariate analysis at both 5% and 25% cutoff values. GPX4 expression was associated with poor OS and DFS, especially in lung adenocarcinoma (p = 0.008, and 0.027, respectively). In conclusions, IHC analysis revealed that GPX4 expression was associated with poor survival outcomes in patients with resected lung adenocarcinoma. Further research is needed to understand the role of GPX4 in tumorigenesis and the underlying mechanism responsible for survival outcomes in patients with resected lung adenocarcinoma.
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Adenocarcinoma del Pulmón , Adenocarcinoma , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Neoplasias Pulmonares/cirugía , Adenocarcinoma del Pulmón/cirugía , Adenocarcinoma/cirugíaRESUMEN
BACKGROUND: Air pollution exposure and idiopathic pulmonary fibrosis (IPF) cause a poor prognosis after SARS-CoV-2 infection, but the underlying mechanisms are not well explored. Angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are the keys to the entry of SARS-CoV-2. We therefore hypothesized that air pollution exposure and IPF may increase the expression of ACE2 and TMPRSS2 in the lung alveolar region. We measured their expression levels in lung tissues of control non-IPF and IPF patients, and used murine animal models to study the deterioration of IPF caused by particulate matter (PM) and the molecular pathways involved in the expression of ACE2 and TMPRSS2. RESULTS: In non-IPF patients, cells expressing ACE2 and TMPRSS2 were limited to human alveolar cells. ACE2 and TMPRSS2 were largely upregulated in IPF patients, and were co-expressed by fibroblast specific protein 1 (FSP-1) + lung fibroblasts in human pulmonary fibrotic tissue. In animal models, PM exposure increased the severity of bleomycin-induced pulmonary fibrosis. ACE2 and TMPRSS2 were also expressed in FSP-1+ lung fibroblasts in bleomycin-induced pulmonary fibrosis, and when combined with PM exposure, they were further upregulated. The severity of pulmonary fibrosis and the expression of ACE2 and TMPRSS2 caused by PM exposure were blocked by deletion of KC, a murine homologue of IL-8, or treatment with reparixin, an inhibitor of IL-8 receptors CXCR1/2. CONCLUSIONS: These data suggested that risk of SARS-CoV-2 infection and COVID-19 disease severity increased by air pollution exposure and underlying IPF. It can be mediated through upregulating ACE2 and TMPRSS2 in pulmonary fibroblasts, and prevented by blocking the IL-8/CXCR1/2 pathway.
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Enzima Convertidora de Angiotensina 2/genética , COVID-19/etiología , Fibrosis Pulmonar Idiopática/complicaciones , Material Particulado/toxicidad , SARS-CoV-2 , Serina Endopeptidasas/genética , Enzima Convertidora de Angiotensina 2/fisiología , Animales , Humanos , Interleucina-8/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Alveolos Pulmonares/enzimología , Serina Endopeptidasas/fisiología , Regulación hacia ArribaRESUMEN
Cancer stem cells (CSCs), a subpopulation of cancer cells responsible for tumor initiation and treatment failure, are more susceptible to ferroptosis-inducing agents than bulk cancer cells. However, regulatory pathways controlling ferroptosis, which can selectively induce CSC death, are not fully understood. Here, we demonstrate that the CSCs of esophageal squamous carcinoma cells enriched by spheroid culture have increased intracellular iron levels and lipid peroxidation, thereby increasing exposure to several products of lipid peroxidation, such as MDA and 4-HNE. However, CSCs do not reduce cell viability until glutathione is depleted by erastin treatment. Mechanistic studies revealed that damage from elevated lipid peroxidation is avoided through the activation of Hsp27, which upregulates GPX4 and thereby rescues CSCs from ferroptosis-induced cell death. Our results also revealed a correlation between phospho-Hsp27 and GPX4 expression levels and poor prognosis in patients with esophageal cancer. Together, these data indicate that targeting Hsp27 or GPX4 to block this intrinsic protective mechanism against ferroptosis is a potential treatment strategy for eradicating CSC in esophageal squamous cell carcinoma.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Ferroptosis , Muerte Celular/fisiología , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Peroxidación de Lípido/fisiología , Células Madre Neoplásicas/metabolismo , Fosfolípido Hidroperóxido Glutatión PeroxidasaRESUMEN
BACKGROUND: Recent advancements in cancer biology field suggest that glucose metabolism is a potential target for cancer treatment. However, little if anything is known about the metabolic profile of cancer stem cells (CSCs) and the related underlying mechanisms. METHODS: The metabolic phenotype in lung CSC was first investigated. The role of collagen XVII, a putative stem cell or CSC candidate marker, in regulating metabolic reprogramming in lung CSC was subsequently studied. Through screening the genes involved in glycolysis, we identified the downstream targets of collagen XVII that were involved in metabolic reprogramming of lung CSCs. Collagen XVII and its downstream targets were then used to predict the prognosis of lung cancer patients. RESULTS: We showed that an aberrant upregulation of glycolysis and oxidative phosphorylation in lung CSCs is associated with the maintenance of CSC-like features, since blocking glycolysis and oxidative phosphorylation reduces sphere formation, chemoresistance, and tumorigenicity. We also showed that the Oct4-hexokinase 2 (HK2) pathway activated by collagen XVII-laminin-332 through FAK-PI3K/AKT-GSB3ß/ß-catenin activation induced the upregulation of glycolysis and maintenance of CSC-like features. Finally, we showed that collagen XVII, Oct4, and HK2 could be valuable markers to predict the prognosis of lung cancer patients. CONCULSIONS: These data suggest the Oct4-HK2 pathway regulated by collagen XVII plays an important role in metabolic reprogramming and maintenance of CSC-like features in lung CSCs, which may aid in the development of new strategies in cancer treatment.
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Autoantígenos/biosíntesis , Reprogramación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/metabolismo , Colágenos no Fibrilares/biosíntesis , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Células A549 , Células HT29 , Humanos , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , Células Madre Pluripotentes/patología , Colágeno Tipo XVIIRESUMEN
Tumor progression with chemoresistance and local recurrence is commonly happened during treatment of esophageal squamous cell carcinoma (ESCC). Cancer stem cells (CSC) may respond for tumor progression. However, there are few reports regarding metabolism of esophageal CSCs with clinical correlation. In this work, we demonstrated that ESCC cell lines in spheroid culture display CSC phenotypes, including increased ALDH activity, chemoresistance and tumor initiation, which are dependent on Hsp27 activation. Esophageal CSCs also exhibit reprogrammed metabolic features particularly higher glycolysis and oxidative phosphorylation, which are regulated via the Hsp27-AKT-HK2 pathway. Moreover, HK2 is required for maintenance of CSC phenotypes. Inhibition of CSC metabolism reduces cell growth and tumor formation. Clinically, patients who underwent surgical resection for esophageal cancer, and displayed overexpression of both Hsp27 and HK2, had the worst prognosis of all expression types. In conclusion, stem cells features and aberrant metabolic reprogramming of esophageal CSCs depend on the Hsp27-AKT-HK2 pathway. Targeting Hsp27 and HK2 could be novel therapeutic strategy for treating esophageal cancer and warrants further investigation.
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Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Choque Térmico/metabolismo , Hexoquinasa/metabolismo , Chaperonas Moleculares/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Desoxiglucosa/farmacología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Hexoquinasa/genética , Humanos , Estimación de Kaplan-Meier , Metformina/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Although particular matter (PM) increases incidence and severity of idiopathic pulmonary fibrosis, the underlying mechanism remains elusive. METHODS: The effects of PM were evaluated in a murine model of bleomycin-induced pulmonary fibrosis. Mice were divided into four groups, receiving: (1) Saline (control), (2) bleomycin, (3) PM, or (4) bleomycin plus PM (Bleo+PM). Additional groups of Bleo+PM mice were treated with sivelestat (an inhibitor of neutrophil elastase) or reparixin (a C-X-C motif chemokine receptor 2 antagonist), or were genetically modified with keratinocyte chemoattractant (KC) deletion. RESULTS: Pulmonary fibrosis was not observed in the control or PM groups. Bleomycin induced pulmonary fibrosis within 14 days. The Bleo+PM group showed worse pulmonary fibrosis when compared to the bleomycin group. Analyses of immune cell profile and chemokine/cytokine concentrations at day 2-bronchoalveolar lavage fluid (BALF) revealed that the Bleo+PM group had increased neutrophil number and elastase level and KC concentration compared to the bleomycin group. Neutrophil elastase activated the Smad2/Smad3/α-SMA pathway to induce collagen deposition, while sivelestat abrogated the increased severity of pulmonary fibrosis caused by PM. Chemotaxis assay revealed that BALF of the Bleo+PM group recruited neutrophil, which was dependent on KC. Further, genetic KC deletion or pharmaceutical inhibition of KC binding to CXCR2 with reparixin ameliorated the PM-induced increased severity of pulmonary fibrosis. CONCLUSIONS: These data provide evidence that the PM-induced increased severity of pulmonary fibrosis depends on KC-mediated neutrophil chemotaxis and give additional mechanic insight that will aid in the development of therapeutic strategies.
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Quimiocina CXCL1/metabolismo , Quimiotaxis , Neutrófilos/efectos de los fármacos , Material Particulado/toxicidad , Fibrosis Pulmonar/etiología , Actinas/genética , Actinas/metabolismo , Animales , Bleomicina/toxicidad , Células Cultivadas , Quimiocina CXCL1/genética , Colágeno/genética , Colágeno/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Neutrófilos/fisiología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Proteínas Smad/genética , Proteínas Smad/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Epithelial-to-mesenchymal transition (EMT) is associated with tumor metastasis and tumorigenesis in lung cancer stem-like cells (CSCs). However, the exact mechanism underlying this is not clear. We used microarray analysis to identify candidate genes responsible for EMT in spheroid and monolayer cultures of lung cancer cells. We found increased expression of a variety of adhesion molecules in CSCs. One of these molecules, Collagen XVII (Col XVII), was demonstrated to be required for maintenance of EMT phenotypes and metastasis ability in lung CSCs. We showed that Col XVII stabilized laminin-5 to activate the FAK/AKT/GSK3ß pathway, thereby suppressing Snail ubiquitination-degradation. The function of Col XVII was mainly dependent on shedding by ADAM9 and ADAM10. Patients who underwent surgical resection for lung cancer, and displayed overexpression of both Col XVII and laminin-5, had the worst prognosis of all expression types. Moreover, blockage of the Col XVII/laminin-5 pathway reduced the EMT phenotypes of lung CSCs in vitro and decreased the potential of lung metastasis in vivo. Our findings suggested that targeting Col XVII and laminin-5 could be novel therapeutic strategies for treating lung cancer patients, and warrant further investigation.
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Pulmonary fibrosis is characterized by fibroblast proliferation and extracellular matrix remodelling, leading to respiratory insufficiency. The mechanisms underlying this progressive and devastating disease remain unclear. Conditions that can impair the function of the endoplasmic reticulum (ER) cause accumulation of unfolded or misfolded proteins, resulting in ER stress and activation of the unfolded protein response (UPR). ER stress has been implicated in many conditions including cancer, diabetes, obesity, and inflammation. It is also involved in lung fibrosis, through myofibroblastic differentiation of fibroblasts; however, the precise role of ER stress in lung fibrosis is unknown. The current study aimed to investigate the underlying mechanisms of ER stress inhibitors in the treatment of bleomycin-induced lung fibrosis. We demonstrated that bleomycin can activate ER stress associated proteins, including GRP78, CHOP, and ATF-4, both in vitro and in vivo. PI3K/AKT acts upstream of ER stress to affect lung fibroblast proliferation, resulting in bleomycin-induced pulmonary fibrosis. Treatment with ER stress inhibitors or a PI3K inhibitor caused a reduction in fibroblast proliferation and improved pulmonary function. The relationship between PI3K/AKT/mTOR and ER stress in pulmonary fibrosis, and the application of PI3K inhibitors and ER stress inhibitors in the treatment of pulmonary fibrosis require further investigation.
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Bleomicina/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Factor de Transcripción Activador 4/metabolismo , Animales , Línea Celular , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Proteínas de Choque Térmico/metabolismo , Pulmón/patología , Ratones , Fibrosis Pulmonar/metabolismo , Factor de Transcripción CHOP/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
UNLABELLED: Epstein-Barr virus (EBV), an oncogenic herpesvirus, has the potential to immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro. During immortalization, several EBV products induce cytokines or chemokines, and most of these are required for the proliferation of LCLs. Interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, is upregulated after EBV infection, and this upregulation is detectable in all LCLs tested. EBV latent membrane protein 1 (LMP1) is responsible for inducing IL-32 expression at the mRNA and protein levels. Mechanistically, we showed that this LMP1 induction is provided by the p65 subunit of NF-κB, which binds to and activates the IL-32 promoter. Furthermore, the short hairpin RNA (shRNA)-mediated depletion of endogenous LMP1 and p65 in LCLs suppressed IL-32 expression, further suggesting that LMP1 is the key factor that stimulates IL-32 in LCLs via the NF-κB p65 pathway. Functionally, knockdown of IL-32 in LCLs elicits viral reactivation and affects cytokine expression, but it has no impact on cell proliferation and apoptosis. Of note, we reveal the mechanism whereby IL-32 is involved in the maintenance of EBV viral latency by inactivation of Zta promoter activity. This atypical cytoplasmic IL-32 hijacks the Zta activator protein kinase Cδ (PKCδ) and inhibits its translocation from the cytoplasm to the nucleus, where PKCδ binds to the Zta promoter and activates lytic cycle progression. These novel findings reveal that IL-32 is involved in the maintenance of EBV latency in LCLs. This finding may provide new information to explain how EBV maintains latency, in addition to viral chromatin structure and epigenetic modification. IMPORTANCE: EBV persists in two states, latency and lytic replication, which is a unique characteristic of human infections. So far, little is known about how herpesviruses maintain latency in particular tissues or cell types. EBV is an excellent model to study this question because more than 90% of people are latently infected. EBV can immortalize primary B cells into lymphoblastoid cell lines in vitro. Expression of IL-32, a novel atypical cytoplasmic proinflammatory cytokine, increased after infection. The expression of IL-32 was controlled by LMP1. In investigating the regulatory mechanism, we demonstrated that the p65 subunit of NF-κB is required for this upregulation. Of note, the important biological activity of IL-32 was to trap protein kinase Cδ in the cytoplasm and prevent it from binding to the Zta promoter, which is the key event for EBV reaction. So, the expression of LMP1-induced IL-32 plays a role in the maintenance of EBV latency.
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Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Interleucinas/biosíntesis , Proteína Quinasa C-delta/metabolismo , Proteínas de la Matriz Viral/metabolismo , Latencia del Virus , Linfocitos B/virología , Células Cultivadas , Herpesviridae , Humanos , Factor de Transcripción ReIA/metabolismoRESUMEN
Epstein Barr virus (EBV) uses various strategies to manipulate host cytokine production in favor of the survival of infected B-cells. Microarray and cytokine protein array assays revealed that tissue inhibitor of metalloproteinase-1 (TIMP-1) was significantly up-regulated in EBV-infected primary B cells and maintained in abundance in EBV-immortalized lymphoblastoid cell lines (LCLs). TIMP-1 plays critical roles in extracellular matrix homeostasis and regulates signaling pathways. In this study, we demonstrated that the EBV-encoded immediate early lytic protein, Zta, upregulates mainly TIMP-1 expression by binding to the AP-1 site within the TIMP-1 promoter. Moreover, knockdown of TIMP-1 expression promoted cisplastin and cold shock-induced death of LCLs. This study provides a mechanistic link between EBV-induced TIMP-1 expression and its impact on LCL survival.
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Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/fisiopatología , Herpesvirus Humano 4/fisiología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Linfocitos B/metabolismo , Linfocitos B/virología , Supervivencia Celular , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Oncogenic Epstein-Barr virus (EBV) uses various approaches to escape host immune responses and persist in B cells. Such persistent infections may provide the opportunity for this virus to initiate tumor formation. Using EBV-immortalized lymphoblastoid cell lines (LCLs) as a model, we found that the expression of major histocompatibility complex (MHC) class II and CD74 in B cells is repressed after EBV infection. Class II transactivator (CIITA) is the master regulator of MHC class II-related genes. As expected, CIITA was downregulated in LCLs. We showed that downregulation of CIITA is caused by EBV latent membrane protein 2A (LMP2A) and driven by the CIITA-PIII promoter. Furthermore, we demonstrated that LMP2A-mediated E47 and PU.1 reduction resulted in CIITA suppression. Mechanistically, the LMP2A immunoreceptor tyrosine-based activation motif was critical for the repression of E47 and PU.1 promoter activity via Syk, Src, and the phosphatidylinositol 3-kinase/Akt pathway. Elimination of LMP2A in LCLs using a shLMP2A approach showed that the expression levels of E47, PU.1, CIITA, MHC class II, and CD74 are reversed. These data indicated that the LMP2A may reduce MHC class II expression through interference with the E47/PU.1-CIITA pathway. Finally, we demonstrated that MHC class II may be detected in tonsils and EBV-negative Hodgkin disease but not in EBV-associated posttransplant lymphoproliferative disease and Hodgkin disease.
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Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/química , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción 3/genética , Proteínas de la Matriz Viral/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Western Blotting , Células Cultivadas , Regulación hacia Abajo , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Humanos , Técnicas para Inmunoenzimas , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción 3/metabolismo , Activación Transcripcional , Proteínas de la Matriz Viral/genéticaRESUMEN
Tumors are influenced by a microenvironment rich in inflammatory cytokines, growth factors and chemokines, which may promote tumor growth. Interleukin-6 (IL-6) is a multifunctional cytokine and known as a regulator of immune and inflammation responses. IL-6 has also been reported to be associated with tumor progression and chemoresistance in different types of cancers. In our study, we demonstrated that IL-6 enriches the properties of lung cancer stem-like cells in A549 lung cancer cells cultured in spheroid medium. IL-6 also promotes sphere formation and stem-like properties of A549 cells by enhancing cell proliferation. Methylation-specific polymerase chain reaction (PCR) was performed and revealed that IL-6 increased methylation of p53 and p21 in A549 cancer cells. Western blot analysis and quantitative real-time PCR demonstrated that IL-6 increased the expression of DNA methyltransferase 1 (DNMT1) in A549 cells cultured in spheroid medium, but not the expression of DNMT3a or DNMT3b. Knockdown of DNMT1 eliminated IL-6-mediated hypermethylation of cell cycle regulators and enrichment of lung cancer stem-like properties. In conclusion, our study, for the first time, shows that the IL-6/JAK2/STAT3 pathway upregulates DNMT1 and enhances cancer initiation and lung cancer stem cell (CSC) proliferation by downregulation of p53 and p21 resulting from DNA hypermethylation. Upon blockage of the IL-6/JAK2/STAT3 pathway and inhibition of DNMT1, the proliferation of lung CSCs was reduced and their formation of spheres and ability to initiate tumor growth were decreased. These data suggest that targeting of the IL-6/JAK2/STAT3 signaling pathway and DNMT1 may become important strategies for treating lung cancer.
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Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/fisiología , Interleucina-6/fisiología , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Humanos , Janus Quinasa 2/fisiología , Ratones , Factor de Transcripción STAT3/fisiología , Esferoides Celulares , Proteína p53 Supresora de Tumor/fisiología , Regulación hacia ArribaRESUMEN
OBJECTIVES: Little is known about the role of Wnt/ß-catenin in postnatal airway homeostasis and basal cell function. This study aimed to investigate the role of Wnt signaling in the self-renewal of basal cells and the involvement of ß-catenin in tracheal repair after naphthalene-induced injury. METHODS: Mice were treated with naphthalene and injected with 4-hydroxytamoxifen. Injury and repair of the tracheal epithelium after naphthalene-mediated secretory cell depletion was assessed by a immunohistochemical study. The involvement of Wnt and ß-catenin signaling in basal cell proliferation was investigated during in vitro expansion. RESULTS: Immunohistochemical analysis of tracheal epithelium in wild-type mice showed a reduction in the number of Clara cell secretory protein (CCSP+) and forkhead box transcription factor (Fox-J1+) cells on days 2 to 5 after naphthalene-induced injury; this cell population was regenerated by day 10. After flush labeling, bromodeoxyuridine-positive (BrdU+) cells and Ki67+ cells were observed in tracheal epithelium on days 2 to 5 but not on days 10 and 21. Confocal microscopy visualizing K5+ and BrdU+ cells showed that Wnt3a promotes proliferation of K5+ cells. Immunohistochemical analysis of K5+ and CCSP+ in tracheal epithelial cells from wild-type littermate and K5-Cre-mediated ß-catenin knock-out mice showed that on day 3, the number of CCSP+ cells was decreased in all mice. On day 10, CCSP+ cells were present in wild-type littermate mice but absent in conditional knock-out mice. CONCLUSIONS: Basal cells serve as stem cells in the tracheal epithelium, regenerating and maintaining tracheal epithelial cells in a mouse model of tracheal injury. ß-Catenin is required for proliferation and self-renewal of tracheal epithelial cells.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Naftalenos/toxicidad , Regeneración/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Células Madre/efectos de los fármacos , Tráquea/efectos de los fármacos , beta Catenina/metabolismo , Animales , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/patología , Factores de Transcripción Forkhead/metabolismo , Queratina-15 , Queratina-5/genética , Queratina-5/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Células Madre/metabolismo , Células Madre/patología , Factores de Tiempo , Tráquea/metabolismo , Tráquea/patología , Uteroglobina/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/genéticaRESUMEN
Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.
Asunto(s)
Linfocitos B/virología , Proliferación Celular , Quimiocina CCL3/biosíntesis , Quimiocina CCL4/biosíntesis , Herpesvirus Humano 4/patogenicidad , Interacciones Huésped-Patógeno , Proteínas de la Matriz Viral/metabolismo , Linfocitos B/metabolismo , Linfocitos B/fisiología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transducción de SeñalRESUMEN
Epstein-Barr virus (EBV) belongs to the gammaherpesvirus family. To produce infectious progeny, EBV reactivates from latency into the lytic cycle by expressing the determinative lytic transactivator, Zta. In the presence of histone deacetylase inhibitor (HDACi), p53 is a prerequisite for the initiation of the EBV lytic cycle by facilitating the expression of Zta. In this study, a serial mutational analysis of Zta promoter (Zp) indicated an important role for the ZID element in responding to HDACi induction and p53 binds to this ZID element together with Sp1, a universal transcription factor. Abolition of the DNA-binding ability of Sp1 reduces the inducibility of ZID by HDACi and also reduces the amount of p53 binding to ZID. Finally, it was shown that EBV in p53-positive-lymphoblastoid cell lines (LCLs) can enter into the lytic cycle spontaneously; however, knockdown of p53 in LCLs leads to retardation of EBV reactivation.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Regiones Promotoras Genéticas/genética , Factor de Transcripción Sp1/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular , Análisis Mutacional de ADN , Herpesvirus Humano 4/genética , Humanos , Factor de Transcripción Sp1/genética , Transactivadores/genética , Proteína p53 Supresora de Tumor/genética , Activación ViralRESUMEN
BACKGROUND: The role of mesenchymal stem cells (MSCs) and IL-6 in lung cancer has not been well-addressed. We aimed to determine if MSCs can enhance the ability of tumor initiation of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway. MATERIALS AND METHODS: Lung cancer cell lines A549 and CL1-5 were directly or indirectly cocultured with MSCs. Spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. Cells without and with MSCs were injected into NOD/SCID mice. The percentage of tumor formation was determined. The influence of the IL-6/JAK2/STAT3 signaling pathway in cancer cell sphere formation and tumor growth were investigated. RESULTS: A very small number of lung cancer cells, when mixed with otherwise non-tumorigenic MSCs, obtained de novo tumorigenicity when injected subcutaneously and allowed to form a tumor xenograft. Secretion of IL-6 from MSCs increased activation of the JAK2/STAT3 pathway in cancer cells, and enhanced sphere formation and tumor initiation. A reduced capacity of tumor formation of A549 and CL1-5 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 and CL1-5 admixed with MSCs. CONCLUSIONS: Culture of A549 or CL1-5 lung cancer cells with MSCs increased sphere formation, drug resistance, and overexpression of pluripotency markers through activation of the IL-6/JAK2/STAT3 pathway. MSCs enhanced the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Blockade of the IL-6/JAK2/STAT3 pathway attenuated the capability of A549 and CL1-5 cells to form tumors.
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Interleucina-6/fisiología , Janus Quinasa 2/fisiología , Neoplasias Pulmonares/etiología , Células Madre Mesenquimatosas/fisiología , Factor de Transcripción STAT3/fisiología , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patología , Ratones , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
EBV, an oncogenic human herpesvirus, can transform primary B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) through multiple regulatory mechanisms. However, the involvement of protein tyrosine kinases in the infinite proliferation of B cells is not clear. In this study, we performed kinase display assays to investigate this subject and identified a specific cellular target, Recepteur d'Origine Nantais (RON) tyrosine kinase, expressed in LCLs but not in primary B cells. Furthermore, we found that latent membrane protein 1 (LMP1), an important EBV oncogenic protein, enhanced RON expression through its C-terminal activation region-1 (CTAR1) by promoting NF-κB binding to the RON promoter. RON knockdown decreased the proliferation of LCLs, and transfection with RON compensated for the growth inhibition caused by knockdown of LMP1. Immunohistochemical analysis revealed a correlation between LMP1 and RON expression in biopsies from posttransplantation lymphoproliferative disorder (PTLD), suggesting that LMP1-induced RON expression not only is essential for the growth of LCLs but also may contribute to the pathogenesis of EBV-associated PTLD. Our study is the first to reveal the impact of RON on the proliferation of transformed B cells and to suggest that RON may be a novel therapeutic target for EBV-associated lymphoproliferative diseases.