RESUMEN
The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells in vitro. We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. We show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro. We further find that injection of this nanobody into mice implanted with 4T1-12B cells orthotopically in the mammary fat pad inhibits metastasis of tumor cells to lung. These results suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis.
Asunto(s)
Neoplasias Pulmonares , Anticuerpos de Dominio Único , ATPasas de Translocación de Protón Vacuolares , Animales , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Femenino , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Línea Celular Tumoral , Neoplasias de la Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Ratones Endogámicos BALB C , Humanos , Invasividad NeoplásicaRESUMEN
BACKGROUND/AIMS: Motivated by the vacuolar proton pump's importance in cancer, we investigate the effects of proton pump inhibition on breast cancer cell migration and proliferation, F-actin polymerization, lamin A/C, heterochromatin, and ETV7 expressions, nuclear size and shape, and AKT/mTOR signaling. METHODS: Lowly metastatic MCF7 and highly metastatic MDA-MB-231 breast cancer cells were treated with 120 nM of proton pump inhibitor Bafilomycin A1 for 24 hours. Cell migration was studied with wound- scratch assays, ATP levels with a chemiluminescent assay; cell proliferation was quantified by a cell area expansion assay. Nuclear size and shape were determined using DAPI nuclear stain and fluorescence microscopy. The levels of F-actin, lamin A/C, heterochromatin, and ETV7 were quantified using both immunocytochemistry and western blots; p-mTORC1, p-mTORC2, mTOR, p-AKT, and AKT were measured by western blots. RESULTS: We reveal that proton pump inhibition reduces F-actin polymerization, cell migration, proliferation, and increases heterochromatin in both lowly and highly metastatic cells. Surprisingly, Bafilomycin decreases lamin A/C in both cell lines. Inhibition has different effects on ETV7 expression in lowly and highly metastatic cells, as well as nuclear area, perimeter, and circularity. Bafilomycin also significantly decreases p-mTORC1, p-MTORC2, and MTOR expression in both cell lines, whereas it significantly decreases p-AKT in lowly metastatic cells and surprisingly significantly increases p-AKT in highly metastatic cells. Our proton pump inhibition protocol reduces V-ATPase levels (~25%) within three hours. V-ATPase levels vary in time for both control and inhibited cells, and inhibition reduces cellular ATP. CONCLUSION: Proton pumps promote F-actin polymerization and decrease heterochromatin, facilitating invasion. These pumps also upregulate both mTORC1 and mTORC2, thus highlighting the relevance of vacuolar proton pumps as metastatic cancer targets.
Asunto(s)
Actinas , Neoplasias de la Mama , Movimiento Celular , Proliferación Celular , Heterocromatina , Macrólidos , Diana Mecanicista del Complejo 2 de la Rapamicina , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , ATPasas de Translocación de Protón Vacuolares , Humanos , Actinas/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Transducción de Señal/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Macrólidos/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Heterocromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Células MCF-7RESUMEN
Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo. In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-II and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular colocalization of LC3 with Lamp2, a lysosome marker, post IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Coimmunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo. These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. SIGNIFICANCE STATEMENT: This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via vacuolar ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond nonsteroidal anti-inflammatory drugs-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.
Asunto(s)
Indometacina , Lisosomas , Sirolimus , ATPasas de Translocación de Protón Vacuolares , Animales , Indometacina/toxicidad , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Sirolimus/farmacología , Ratones , Masculino , Ratas , Antiinflamatorios no Esteroideos/farmacología , Catepsina B/metabolismo , Ratones Endogámicos C57BL , Línea Celular , Intestino Delgado/efectos de los fármacos , Intestino Delgado/patología , Intestino Delgado/metabolismo , Úlcera/inducido químicamente , Úlcera/patología , Úlcera/metabolismoRESUMEN
Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing.
Asunto(s)
Antivirales , ATPasas de Translocación de Protón Vacuolares , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Ratones , Relación Estructura-Actividad , Antivirales/farmacología , Antivirales/química , Antivirales/farmacocinética , Antivirales/síntesis química , Humanos , Estructura Molecular , Ebolavirus/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Relación Dosis-Respuesta a Droga , Lignanos/farmacología , Lignanos/química , Naftalenos/farmacología , Naftalenos/química , Naftalenos/farmacocinética , Naftalenos/síntesis química , Internalización del Virus/efectos de los fármacosRESUMEN
In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.
Asunto(s)
Endosomas , Fosfohidrolasa PTEN , Fosfatidilinositoles , Vacuolas , Vacuolas/metabolismo , Vacuolas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/efectos de los fármacos , Humanos , Fosfatidilinositoles/metabolismo , Animales , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Ratones , Morfolinas/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética , Citoplasma/metabolismo , Células HeLa , Aminopiridinas , Compuestos Heterocíclicos con 3 AnillosRESUMEN
Background: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing. Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain. Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH. Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.
Asunto(s)
Antivirales , Lignanos , Animales , Humanos , Antivirales/farmacología , Antivirales/química , Antivirales/farmacocinética , Interacciones Hidrofóbicas e Hidrofílicas , Nanopartículas/química , Proteínas Recombinantes/química , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Paclitaxel serves as the cornerstone chemotherapy for ovarian cancer, yet its prolonged administration frequently culminates in drug resistance, presenting a substantial challenge. Here we reported that inducing alkaliptosis, rather than apoptosis or ferroptosis, effectively overcomes paclitaxel resistance. Mechanistically, ATPase H+ transporting V0 subunit D1 (ATP6V0D1), a key regulator of alkaliptosis, plays a pivotal role by mediating the downregulation of ATP-binding cassette subfamily B member 1 (ABCB1), a multidrug resistance protein. Both ATP6V0D1 overexpression through gene transfection and pharmacological enhancement of ATP6V0D1 protein stability using JTC801 effectively inhibit ABCB1 upregulation, resulting in growth inhibition in drug-resistant cells. Additionally, increasing intracellular pH to alkaline (pH 8.5) via sodium hydroxide application suppresses ABCB1 expression, whereas reducing the pH to acidic conditions (pH 6.5) with hydrochloric acid amplifies ABCB1 expression in drug-resistant cells. Collectively, these results indicate a potentially effective therapeutic strategy for targeting paclitaxel-resistant ovarian cancer by inducing ATP6V0D1-dependent alkaliptosis.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Resistencia a Antineoplásicos , Neoplasias Ováricas , Paclitaxel , ATPasas de Translocación de Protón Vacuolares , Humanos , Femenino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Paclitaxel/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Concentración de Iones de Hidrógeno , Proliferación Celular/efectos de los fármacosRESUMEN
Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.
Asunto(s)
Antivirales/farmacología , Lignanos/farmacología , Virus/efectos de los fármacos , Animales , Antígenos Virales/metabolismo , Antivirales/síntesis química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Glucósidos/farmacología , Lignanos/síntesis química , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Virus/clasificación , Virus/metabolismoRESUMEN
Melatonin is a hormone secreted by the pineal gland that is involved in the biorhythm of reproductive activities. The present study investigated the inhibitory effects of melatonin on osteoclastogenesis in RAW 264.7 cells according to changes in V-ATPase and the corresponding inhibition of the MAPK and NFATc1 signaling processes. METHODS: the cytotoxic effect of melatonin was investigated by MTT assay. Osteoclast differentiation and gene expression of osteoclast-related factors were confirmed via TRAP staining, pit formation assay, immunofluorescence imaging, western blot, and real-time PCR. RESULTS: melatonin was found to inactivate the p38 and JNK of MAP kinase in RAW264.7 cells treated with RANKL and treated with a combination RANKL and melatonin for 1, 3, and 5 days. The melatonin treatment group showed a reduction in osteoclastogenesis transcription factors and ATP6v0d2 gene expression. CONCLUSIONS: melatonin inhibits osteoclast differentiation and cell fusion by inhibiting the expression of Atp6v0d2 through the inactivation of MAPK and NFATc1 signaling in RANKL-stimulated RAW264.7 macrophages. The findings of the present study suggest that melatonin could be a suitable therapy for bone loss and imply a potential role of melatonin in bone health.
Asunto(s)
Melatonina/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Factores de Transcripción NFATC/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Osteoclastos/citología , Osteogénesis , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Antioxidantes/farmacología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Células Cultivadas , Regulación hacia Abajo , Regulación de la Expresión Génica , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , FN-kappa B/antagonistas & inhibidores , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Células RAW 264.7RESUMEN
Hepatocellular carcinoma (HCC) has high morbidity and mortality. MicroRNAs (miRNAs), which could be regulated by cancer-derived exosomes, play critical regulatory roles in the initiation and development of cancer. However, the expressions, effects, and mechanisms of abundant miRNAs regulated by HCC cancer-derived exosomes in HCC remain largely unclear. Exosomes of HepG2 cells under heat shock, TGF-ß1, doxorubicin, acid and hypoxia/reoxygenation (H/R) conditions, and exosomes were successfully identified by transmission electron microscopy and Western blot analysis. The identified exosomes were then applied to evaluate the miRNA expression profiles by RNA sequencing. Mechanically, we discovered that doxorubicin was upregulated, TGF-ß1 downregulated the expressions of Vps4A, Rab27A, Alix, and Hrs in HepG2 cells and exosomes, and Vps4A and Rab27A, as target genes for miR-4454, could also be downregulated by miR-4454. Functionally, we revealed that miR-4454 inhibitor and miR-4454 inhibitor-mediated exosomes could markedly suppress proliferation, migration, invasion, and vascularization and accelerate cycle arrest, apoptosis, and ROS of HepG2 cells. This study provided many potential HCC cancer-derived exosome-mediated miRNAs in HCC under 5 different stimulus conditions. Meanwhile, we certified that miR-4454 in exosomes could provide a novel and effective mechanism for HCC function.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/patología , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , MicroARNs/genética , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Proteínas rab27 de Unión a GTP/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimiento Celular , Proliferación Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Células Tumorales Cultivadas , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas rab27 de Unión a GTP/genética , Proteínas rab27 de Unión a GTP/metabolismoRESUMEN
At present, liver ischemia-reperfusion (IR) injury is still a great challenge for clinical liver partial resection and liver transplantation. The innate immunity regulated by liver macrophages orchestrates the cascade of IR inflammation and acts as a bridge. As a specific macrophage subunit of vacuolar ATPase, ATP6V0D2 (V-ATPase D2 subunit) has been shown to promote the formation of autophagolysosome in vitro. Our research fills a gap which has existed in the study of inflammatory stress about the V-ATPase subunit ATP6V0D2 in liver macrophages. We first found that the expression of specific ATP6V0D2 in liver macrophages was upregulated with the induction of inflammatory cascade after liver IR surgery, and knockdown of ATP6V0D2 resulted in increased secretion of proinflammatory factors and chemokines, which enhanced activation of NLRP3 and aggravation of liver injury. Further studies found that the exacerbated activation of NLRP3 was related to the autophagic flux regulated by ATP6V0D2. Knocking down ATP6V0D2 impaired the formation of autophagolysosome and aggravated liver IR injury through nonspecific V-ATPase activation independent of V-ATPase-Notchl-Hesl signal axis. In general, we illustrated that the expression of ATP6V0D2 in liver macrophages was upregulated after liver IR, and by gradually promoting the formation of autophagolysosomes to increase autophagy flux to limit the activation of liver inflammation, this regulation is independent of the Notch1-Hes1 signal axis.
Asunto(s)
Autofagia/inmunología , Inflamasomas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Daño por Reperfusión/inmunología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Autofagosomas/inmunología , Autofagosomas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Hepatectomía/efectos adversos , Hepatectomía/métodos , Humanos , Inflamasomas/metabolismo , Hígado/irrigación sanguínea , Hígado/inmunología , Hígado/patología , Hígado/cirugía , Trasplante de Hígado/efectos adversos , Lisosomas/inmunología , Lisosomas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Receptor Notch1/metabolismo , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Transducción de Señal/inmunología , Factor de Transcripción HES-1/metabolismo , Regulación hacia Arriba , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
In a newborn pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a lower airway surface liquid (ASL) pH. In the gland-containing airways, the ASL pH is balanced by CFTR (CF transmembrane conductance regulator) and ATP12A, which, respectively, control HCO3- transport and proton secretion. We found that, although porcine small airway tissue expressed lower amounts of ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter < 200 µm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that gland-containing airways and small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0 d subunit of the H+-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0 d2 subunit isoform at the apical surface of Muc5B+ secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO3-, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airway diseases.
Asunto(s)
Bicarbonatos/metabolismo , Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Animales Modificados Genéticamente , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Masculino , Porcinos , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.
Asunto(s)
Macrólidos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biocatálisis/efectos de los fármacos , Bovinos , Microscopía por Crioelectrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Macrólidos/química , Macrólidos/metabolismo , Modelos Moleculares , Estructura Molecular , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/ultraestructuraRESUMEN
Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splice-variant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown.Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9-mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform.Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Humanos , Masculino , TransfecciónRESUMEN
Aims: A high-salt diet can aggravate oxidative stress, and renal fibrosis via the brain and renal renin-angiotensin system (RAS) axis in chronic kidney disease (CKD) rats. (Pro)renin receptor (PRR) plays a role in regulating RAS and oxidative stress locally. However, whether central PRR regulates salt-induced renal injury in CKD remains undefined. Here, we hypothesized that the reduction of central PRR expression could ameliorate central lesions and thereby ameliorate renal injury in high-salt-load CKD rats. Results: We investigated RAS, sympathetic nerve activity, oxidative stress, inflammation, and tissue injury in subfornical organs and kidneys in high-salt-load 5/6 nephrectomy CKD rats after the silencing of central PRR expression by intracerebroventricular lentivirus-RNAi. We found that the sympathetic nerve activity was reduced, and the levels of inflammation and oxidative stress were decreased in both brain and kidney. Renal injury and fibrosis were ameliorated. To explore the mechanism by which central inhibition of PRR expression ameliorates kidney damage, we blocked central MAPK/ERK1/2 and PI3K/Akt signaling pathways as well as angiotensin converting enzyme 1-angiotensin II-angiotensin type 1 receptors (ACE1-Ang II-AT1R) axis. Salt-induced overexpression of renal RAS, inflammation, oxidative stress, and fibrosis in CKD rats were prevented by central blockade of the pathways. Innovation: This study provides new insights into the mechanisms underlying salt-induced kidney damage. Targeting central PRR or PRR-mediated signaling pathway may be a novel strategy for the treatment of CKD. Conclusions: These results suggested that the silencing of central PRR expression ameliorates salt-induced renal injury in CKD through Ang II-dependent and -independent pathways.
Asunto(s)
Vectores Genéticos/administración & dosificación , Receptores de Superficie Celular/genética , Insuficiencia Renal Crónica/terapia , Sistema Renina-Angiotensina/efectos de los fármacos , Sodio en la Dieta/efectos adversos , ATPasas de Translocación de Protón Vacuolares/genética , Angiotensina II/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Vectores Genéticos/genética , Infusiones Intraventriculares , Lentivirus/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Nefrectomía/efectos adversos , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Insuficiencia Renal Crónica/inducido químicamente , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/antagonistas & inhibidores , Escherichia coli/enzimología , Lisosomas/enzimología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Línea Celular Transformada , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Escherichia coli/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Homeostasis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/efectos de los fármacos , Macrólidos/farmacología , Transfección/métodosRESUMEN
Callyspongiolide is a marine-derived macrolide that kills cells in a caspase-independent manner. NCI COMPARE analysis of human tumor cell line toxicity data for synthetic callyspongiolide indicated that its pattern of cytotoxicity correlated with that seen for concanamycin A, an inhibitor of the vacuolar-type H+-ATPase (V-ATPase). Using yeast as a model system, we report that treatment with synthetic callyspongiolide phenocopied a loss of V-ATPase activity including (1) inability to grow on a nonfermentable carbon source, (2) rescue of cell growth via supplementation with Fe2+, (3) pH-sensitive growth, and (4) a vacuolar acidification defect visualized using the fluorescent dye quinacrine. Crucially, in an in vitro assay, callyspongiolide was found to dose-dependently inhibit yeast V-ATPase (IC50 = 10 nM). Together, these data identify callyspongiolide as a new and highly potent V-ATPase inhibitor. Notably, callyspongiolide is the first V-ATPase inhibitor known to be expelled by Pdr5p.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Macrólidos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/química , Humanos , Concentración de Iones de Hidrógeno , Macrólidos/química , Estructura Molecular , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar-ATPase (V-ATPase) proton pumps encompass unique plasma membrane-associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the in vivo therapeutic efficacy of an antibody (a2v-mAB) targeting specific V-ATPase-'V0a2' surface isoform in controlling ovarian tumor growth. In vitro a2v-mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. In vivo intraperitoneal a2v-mAb treatment drastically delayed ovarian tumor growth with no measurable in vivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v-mAb-treated tumor tissues displayed high immune cell infiltration (M1-macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN-y, IL-1α) compared to control. There was marked decrease in CA-125-positive cancer cells and an enhanced active caspase-3 expression in a2v-mAb-treated tumors. RNA-seq analysis of a2v-mAb tumor tissues further revealed upregulation of apoptosis-related and toll-like receptor pathway-related genes. Indirect coculture of a2v-mAb-treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN-y, IL-17, and IL-12-A in PBMCs, further validating the in vivo antitumor responses. In conclusion, V-ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Inmunidad , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Caspasa 3/metabolismo , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo Condicionados/farmacología , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Desnudos , Clasificación del Tumor , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores Toll-Like/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
We previously reported that silencing of the PRR gene, which encodes the (pro)renin receptor [(P)RR], significantly reduced Wnt/ß-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC). Here, we examined the effects of a panel of blocking mAbs directed against the (P)RR extracellular domain on proliferation of the human PDAC cell lines PK-1 and PANC-1 in vitro and in vivo We observed that four rat anti-(P)RR mAbs induced accumulation of cells in the G0-G1-phase of the cell cycle and significantly reduced proliferation in vitro concomitant with an attenuation of Wnt/ß-catenin signaling. Systemic administration of the anti-(P)RR mAbs to nude mice bearing subcutaneous PK-1 xenografts significantly decreased tumor expression of active ß-catenin and the proliferation marker Ki-67, and reduced tumor growth. In contrast, treatment with the handle region peptide of (pro)renin did not inhibit tumor growth in vitro or in vivo, indicating that the effects of the anti-(P)RR mAbs were independent of the renin-angiotensin system. These data indicate that mAbs against human (P)RR can suppress PDAC cell proliferation by hindering activation of the Wnt/ß-catenin signaling pathway. Thus, mAb-mediated (P)RR blockade could be an attractive therapeutic strategy for PDAC.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores de Superficie Celular/química , ATPasas de Translocación de Protón Vacuolares/química , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Carcinoma Ductal Pancreático/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones , Neoplasias Pancreáticas/metabolismo , Dominios Proteicos , Ratas , Receptores de Superficie Celular/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Vía de Señalización Wnt/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multidrug resistance (MDR) in cancer remains a major challenge for the success of chemotherapy. Natural products have been a rich source for the discovery of drugs against MDR cancers. Here, we applied high-throughput cytotoxicity screening of an in-house natural product library against MDR SGC7901/VCR cells and identified that the cyclodepsipeptide verucopeptin demonstrated notable antitumor potency. Cytological profiling combined with click chemistry-based proteomics revealed that ATP6V1G directly interacted with verucopeptin. ATP6V1G, a subunit of the vacuolar H+-ATPase (v-ATPase) that has not been previously targeted, was essential for SGC7901/VCR cell growth. Verucopeptin exhibited strong inhibition of both v-ATPase activity and mTORC1 signaling, leading to substantial pharmacological efficacy against SGC7901/VCR cell proliferation and tumor growth in vivo. Our results demonstrate that targeting v-ATPase via its V1G subunit constitutes a unique approach for modulating v-ATPase and mTORC1 signaling with great potential for the development of therapeutics against MDR cancers.