RESUMO
Background: Pancreatic adenocarcinoma (PAAD) is a formidable challenge in oncology research, with a complex pathogenesis that requires to be explored. Major Vault Protein (MVP) is the principal structural component of the vault complex, and its expression level is remarkably upregulated in various cancers. Extensive investigations have been conducted to explore the role of MVP in specific cancer contexts, yet the potential molecular mechanisms and biological functions of MVP in PAAD still remain considerably elusive. This study aims to explore the role of MVP as a novel immune-related biomarker in the pathogenesis and clinical treatment of PAAD. Methods: Gene expression data and clinical information were collected from TCGA, GTEx and GEO databases. Survival, prognostic and functional enrichment analysis were employed with R software. Immunological correlation analysis was performed using TIMER2.0, TIDE scores, TISIDB and TISCH. Epigenetic analysis was implemented by MethSurv, CPTAC, UALCAN, and cBioPortal. Drug analysis was conducted using Enrichr and CellMiner. Moreover, cellular experiments, like RNA interference, qRT-PCR, Western blot, cell cycle analysis, cell apoptosis analysis, colony formation assay, transwell assay, and wound healing assay, were performed for verifying the functional properties of MVP in the PAAD progression. Results: We demonstrated an abnormally upregulated expression of MVP in PAAD tissues, which notably correlated with an adverse prognosis in PAAD patients. Functional analysis suggested the conceivable involvement of MVP in immune modulation, and immunotherapy. Additionally, we identified genetic alterations, reduced promoter methylation, and heightened phosphorylation in MVP. We also clarified Suloctidil and Tetradioxin as the most notable potential drugs targeting MVP in PAAD. Moreover, our experimental observations consistently highlighted the significant impact of MVP deficiency on impeding PAAD cell proliferation, inhibiting cell migration, and accelerating cell apoptosis. Interestingly, a potential link between MVP and ERK or AKT pathways was displayed, which opens new avenues for further exploration of the molecular mechanisms of MVP-targeted therapies in PAAD. Conclusions: This study systematically describes MVP as an immune-related biomarker with remarkable potential for predicting the prognosis, tumor progression and immunotherapeutic efficacy in PAAD.
Assuntos
Adenocarcinoma , Biomarcadores Tumorais , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas , Partículas de Ribonucleoproteínas em Forma de Abóbada , Humanos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Prognóstico , ApoptoseRESUMO
Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.
Assuntos
Partículas de Ribonucleoproteínas em Forma de Abóbada , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Humanos , Animais , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/químicaRESUMO
As we previously revealed, major vault protein (MVP) is a virus-induced host factor, and its expression is crucial for innate immune responses. Nevertheless, the function of MVP in adaptive immunity is poorly known. Here, we demonstrate that Mvp knockout mice had attenuated antibody responses and reduced survival after rechallenge with homologous influenza A virus (IAV) relative to wild-type mice. Analysis of B cell populations showed that MVP promoted germinal center (GC) responses to develop optimal antiviral humoral immunity. Although MVP-deficient T cells and dendritic cells (DCs) were not intrinsically damaged, MVP promoted activating effector T cells and T follicular helper responses and regulated specific DC subsets. These findings suggest that MVP directs an effective adaptive immune response against IAV by directly engaging in GC reactions or indirectly augmenting cellular immunity via innate immune pathways.
Assuntos
Imunidade Adaptativa , Células Dendríticas , Imunidade Inata , Vírus da Influenza A , Camundongos Knockout , Infecções por Orthomyxoviridae , Partículas de Ribonucleoproteínas em Forma de Abóbada , Animais , Vírus da Influenza A/imunologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/imunologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Células Dendríticas/imunologia , Camundongos Endogâmicos C57BL , Centro Germinativo/imunologia , Linfócitos B/imunologiaRESUMO
Major vault protein (MVP) is the main component of the vault complex, which is a highly conserved ribonucleoprotein complex found in most eukaryotic organisms. MVP or vaults have previously been found to be overexpressed in multidrug-resistant cancer cells and implicated in various cellular processes such as cell signaling and innate immunity. The precise function of MVP is, however, poorly understood and its expression and probable function in lower eukaryotes are not well characterized. In this study, we report that the Atlantic salmon louse expresses three full-length MVP paralogues (LsMVP1-3). Furthermore, we extended our search and identified MVP orthologues in several other ecdysozoan species. LsMVPs were shown to be expressed in various tissues at both transcript and protein levels. In addition, evidence for LsMVP to assemble into vaults was demonstrated by performing differential centrifugation. LsMVP was found to be highly expressed in cement, an extracellular material produced by a pair of cement glands in the adult female salmon louse. Cement is important for the formation of egg strings that serve as protective coats for developing embryos. Our results imply a possible novel function of LsMVP as a secretory cement protein. LsMVP may play a role in structural or reproductive functions, although this has to be further investigated.
Assuntos
Copépodes , Partículas de Ribonucleoproteínas em Forma de Abóbada , Animais , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Copépodes/metabolismo , Salmo salar/parasitologia , Salmo salar/metabolismo , Feminino , Filogenia , Sequência de AminoácidosRESUMO
Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody's Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z's applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.
Assuntos
Nanopartículas , Trastuzumab , Partículas de Ribonucleoproteínas em Forma de Abóbada , Humanos , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Nanopartículas/química , Trastuzumab/química , Linhagem Celular Tumoral , Cetuximab/química , Anticorpos Monoclonais/química , Imunoconjugados/químicaRESUMO
Major Vault Protein (MVP) has emerged as a potential prognostic and immunological biomarker in various cancer types. This pan-cancer study aimed to investigate expression of MVP and its correlation with clinical outcomes and immune infiltration across diverse cancer types. We conducted an analysis of extensive transcriptomic and clinical data from publicly available databases. Our findings unveiled a significant association between MVP expression and cancer progression, with higher expression levels predicting poorer overall survival in multiple cancer types. Importantly, MVP expression demonstrated a close relationship with immune infiltration in the tumor microenvironment, showing that higher expression levels were associated with increased immune cell infiltration. We further validated expression of MVP and function in cancer cell lines A549 and AGS. These compelling results suggest that MVP holds promise as a valuable biomarker for prognostic assessment and the development of immunotherapeutic strategies across various cancer types. Consequently, targeting MVP may offer a compelling therapeutic approach in the treatment of human cancers.
Assuntos
Biomarcadores Tumorais , Neoplasias , Microambiente Tumoral , Partículas de Ribonucleoproteínas em Forma de Abóbada , Humanos , Neoplasias/imunologia , Neoplasias/mortalidade , Prognóstico , Microambiente Tumoral/imunologia , Biomarcadores Tumorais/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular TumoralRESUMO
OBJECTIVE: Major vault protein (MVP) is vital in various macrophage-related inflammatory diseases. However, the effects of MVP on macrophage polarization during fracture repair are still unknown. METHODS: We used Mvpflox/floxLyz2-Cre mice (myeloid-specific MVP gene knockout, abbreviated as MacKO) and Mvpflox/flox (abbreviated as MacWT) mice to compare their fracture healing phenotype. Next, we traced the changes in macrophage immune status in vivo and in vitro. We further explored the effects of MVP on osteogenesis and osteoclastogenesis. Finally, we re-expressed MVP in MacKO mice to confirm the role of MVP in fracture healing. RESULTS: The lack of MVP in macrophages impaired their transition from a pro-inflammatory to an anti-inflammatory phenotype during fracture repair. The increased secretion of pro-inflammatory cytokines by macrophages promoted their osteoclastic differentiation and impaired BMSC osteogenic differentiation, ultimately leading to impaired fracture repair in MacKO mice. Last, adeno-associated virus (AAV)-Mvp tibial injection significantly promoted fracture repair in MacKO mice. CONCLUSIONS: Our findings showed MVP has a previously unknown immunomodulatory role in macrophages during fracture repair. Targeting macrophage MVP may represent a novel therapeutic method for fracture treatment.
Assuntos
Macrófagos , Osteogênese , Camundongos , Animais , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/farmacologia , Citocinas/metabolismoRESUMO
Tumor-associated macrophages (TAMs) are critical in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC). Major vault protein (MVP) mediates multidrug resistance, cell growth and development, and viral immunity. However, the relationship between MVP and TAMs polarization has not been clarified in HCC. We found that MVP significantly increased M2-TAMs infiltration levels in tumor tissues of HCC patients. MVP promoted HCC proliferation, metastasis, and invasion by regulating M2 polarization in vivo and in vitro. Mechanistically, MVP associated with signal transducer and activator of transcription 6 (STAT6) and enhanced STAT6 phosphorylation. STAT6 translocated from the cytosol to the nucleus and regulated M2 macrophage-associated gene transcription. These findings suggest that MVP modulates the macrophage M2 transcriptional program, revealing its potential role in the TAMs of TME.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Fator de Transcrição STAT6 , Partículas de Ribonucleoproteínas em Forma de Abóbada , Humanos , Fator de Transcrição STAT6/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
Distant metastasis is the primary cause of breast cancer-associated death. The existing information, such as the precise molecular mechanisms and effective therapeutic strategies targeting metastasis, is insufficient to combat breast cancer. This study demonstrates that the protein tyrosine phosphatase PTPN18 is downregulated in metastatic breast cancer tissues and is associated with better metastasis-free survival. Ectopic expression of PTPN18 inhibits breast cancer cell metastasis. PTPN18 is translocated from the cytoplasm to the nucleus by MVP and importin ß2 in breast cancer. Then, nuclear PTPN18 dephosphorylates ETS1 and promotes its degradation. Moreover, nuclear PTPN18 but not cytoplasmic PTPN18 suppresses transforming growth factor-ß signaling and epithelial-to-mesenchymal transition by targeting ETS1. Our data highlight PTPN18 as a suppressor of breast cancer metastasis and provide an effective antimetastatic therapeutic strategy.
Assuntos
Neoplasias da Mama , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Transporte Ativo do Núcleo Celular , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Metástase Neoplásica , Proteínas Tirosina Fosfatases não Receptoras/metabolismoRESUMO
BACKGROUND: Macrophages are implicated in atherosclerotic plaque instability by inflammation and degradation of extracellular matrix. However, the regulatory mechanisms driving these macrophage-associated processes are not well understood. Here, we aimed to identify the plaque destabilization-associated cytokines and signaling pathways in macrophages. METHODS: The atherosclerotic models of myeloid-specific MVP (major vault protein) knockout mice and control mice were generated. Atherosclerotic instability, macrophage inflammatory signaling, and active cytokines released by macrophages were examined in vivo and in vitro by using cellular and molecular biological approaches. RESULTS: MVP deficiency in myeloid cells exacerbated murine plaque instability by increasing production of both MMP (matrix metallopeptidase)-9 and proinflammatory cytokines in artery wall. Mechanistically, expression of MMP-9 was mediated via ASK1 (apoptosis signal-regulating kinase 1)-MKK-4 (mitogen-activated protein kinase kinase 4)-JNK (c-Jun N-terminal kinase) signaling in macrophages. MVP and its α-helical domain could bind with ASK1 and inhibit its dimerization and phosphorylation. A 62 amino acid peptide (MVP-[686-747]) in the α-helical domain of MVP showed a crucial role in preventing macrophage MMP-9 production and plaque instability. CONCLUSIONS: MVP may act as an inhibitor for ASK1-JNK signaling-mediated MMP-9 production in macrophages and, thereby, attenuate unstable plaque formation. Our findings suggest that suppression of macrophage ASK1-JNK signaling may be a useful strategy antagonizing atherosclerotic diseases.
Assuntos
Aterosclerose , Placa Aterosclerótica , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Citocinas/metabolismo , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Placa Aterosclerótica/metabolismo , Partículas de Ribonucleoproteínas em Forma de AbóbadaRESUMO
BACKGROUND: Doxorubicin (DOX) has limited chemotherapy application for malignancies due to cardiotoxicity. The pathogenesis of DOX-induced cardiomyopathy (DiCM) is yet to be elucidated. Increasing studies proved that activation of AKT prevented cardiomyocyte apoptosis and cardiac dysfunction in response to DOX insult. Our previous studies indicated that major vault protein (MVP) deficiency was accompanied by suppressed phosphorylation of AKT in metabolic diseases. This study aimed to investigate the role and underlying mechanism of MVP on cardiomyocyte apoptosis in DiCM. METHODS: Mice were intraperitoneally injected with DOX 5 mg/kg, once a week for 5 weeks, the total cumulative dose was 25 mg/kg. Cardiomyocyte-specific MVP overexpression was achieved using an adeno-associated virus system under the cTnT promoter after the fourth DOX injection. Cardiac function was examined by echocardiography followed by euthanasia. Tissue and serum were collected for morphology analysis and biochemical examination. RESULTS: Herein, we found that MVP expression was upregulated in DOX-treated murine hearts. Cardiac-specific MVP overexpression alleviated DOX-induced cardiac dysfunction, oxidative stress and fibrosis. Mechanistically, MVP overexpression activated AKT signaling and decreased cardiomyocyte apoptosis in DiCM. CONCLUSIONS: Based on these findings, we supposed that MVP was a potential therapeutic agent against DiCM.
Assuntos
Cardiomiopatias , Cardiopatias , Animais , Apoptose , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/prevenção & controle , Cardiotoxicidade/etiologia , Cardiotoxicidade/metabolismo , Cardiotoxicidade/prevenção & controle , Doxorrubicina/efeitos adversos , Cardiopatias/complicações , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Partículas de Ribonucleoproteínas em Forma de AbóbadaRESUMO
Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault's components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.
Assuntos
Neoplasias Colorretais/genética , Metástase Neoplásica/genética , Poli(ADP-Ribose) Polimerases/genética , Proteínas de Ligação a RNA/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Adulto , Idoso , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/patologia , Transcriptoma/genéticaRESUMO
Porcine reproductive and respiratory syndrome (PRRS), a significant viral infectious disease that commonly occurs among farmed pigs, leads to considerable economic losses to the swine industry worldwide. Major vault protein (MVP) is a host factor that induces type â interferon (IFN) production. In this study, we evaluated the effect of MVP on PRRSV infection in CRL2843CD163 cell lines and porcine alveolar macrophages (PAMs). Our results showed that MVP expression was downregulated by PRRSV infection. Adenoviral overexpression of MVP inhibited PRRSV replication, whereas the siRNA knockdown of MVP promoted PRRSV replication. In addition, MVP knockdown has an adverse effect on the inhibitive role of MVP overexpression on PRRSV replication. Moreover, MVP could induce the expression of type â IFNs and IFN-stimulated gene 15 (ISG15) in PRRSV-infected PAMs. Based on these results, MVP may be a potential molecular target of drugs for the effective prevention and treatment of PRRSV infection.
Assuntos
Macrófagos Alveolares/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Animais , Linhagem Celular , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Macrófagos Alveolares/metabolismo , Suínos , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Replicação ViralRESUMO
Rationale: Bone homeostasis is maintained by a balanced interplay of osteoblasts and osteoclasts. Osteoclasts are derived from monocyte/macrophage lineage. Major vault protein (MVP) is known to promote apoptosis and prevent metabolic diseases in macrophage. However, whether MVP is involved in osteoclastogenesis is unknown. Here, we identified an important function of MVP as a negative regulator of osteoclastogenesis and its therapeutic potential in preventing bone loss. Methods: Expression of MVP in osteoclasts was investigated in human tumor tissues with immunohistochemical staining. Next, we generated total body (Mvp-/- ) and monocyte-specific (Mvpf/fLyz2-Cre) MVP gene knockout mice to observe bone phenotype and osteoclastogenesis using micro-CT and bone histomorphometry. Moreover, we examined the effects of MVP on osteoclast differentiation, bone resorption, NFATc1 activation and calcium oscillations in vitro. Finally, we explored the clinical potential of targeting MVP in two osteoporosis mouse models and used an adeno-associated virus (AAV) gene to overexpress MVP locally in mice. Results: We found that Mvp-/- and Mvpf/fLyz2-Cre mice both exhibited osteoporosis-like phenotypes. MVP-deficiency also enhanced calcineurin-NFATc1 signaling and promoted NFATc1 activity, which led to enhanced osteoclastogenesis and bone resorption. Calcineurin inhibition using the small molecule inhibitor FK506 corrected the enhanced osteoclastogenesis in Mvpf/fLyz2-Cre group. Additionally, MVP reexpression in Mvpf/fLyz2-Cre group rescued calcineurin expression. MVP overexpression in wild-type mice prevented pathologic bone loss in mouse models of ovariectomized (OVX) and calvaria-adjacent lipopolysaccharide (LPS)-injected. Conclusions: Our data suggested that MVP negatively regulates osteoclast differentiation and bone resorption via inhibition of calcineurin-NFATc1 signaling. In osteoclast-related bone diseases such as osteoporosis, manipulation of MVP activity may be an attractive therapeutic target.
Assuntos
Calcineurina/metabolismo , Diferenciação Celular , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Calcineurina/genética , Humanos , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
BACKGROUND: Polyamidoamine (PAMAM) dendrimer applications have extended from tumor cells to multidrug-resistant tumor cells. However, their transportation in multidrug-resistant tumor cells remains unclear. Herein, we investigated the exocytosis rule and mechanism of PAMAM dendrimers in multidrug-resistant tumor cells. RESULTS: Using a multidrug-resistant human breast cancer cell model (MCF-7/ADR), we performed systematic analyses of the cellular exocytosis dynamics, pathways and mechanisms of three PAMAM dendrimers with different surface charges: positively charged PAMAM-NH2, neutral PAMAM-OH and negatively charged PAMAM-COOH. The experimental data indicated that in MCF-7/ADR cells, the exocytosis rate was the highest for PAMAM-NH2 and the lowest for PAMAM-OH. Three intracellular transportation processes and P-glycoprotein (P-gp) participated in PAMAM-NH2 exocytosis in MCF-7/ADR cells. Two intracellular transportation processes, P-gp and multidrug resistance (MDR)-associated protein participated in PAMAM-COOH exocytosis. P-gp and MDR-associated protein participated in PAMAM-OH exocytosis. Intracellular transportation processes, rather than P-gp and MDR-associated protein, played major roles in PAMAM dendrimer exocytosis. PAMAM-NH2 could enter MCF-7/ADR cells by forming nanoscale membrane holes, but this portion of PAMAM-NH2 was eliminated by P-gp. Compared with PAMAM-OH and PAMAM-COOH, positively charged PAMAM-NH2 was preferentially attracted to the mitochondria and cell nuclei. Major vault protein (MVP) promoted exocytosis of PAMAM-NH2 from the nucleus but had no effect on the exocytosis of PAMAM-OH or PAMAM-COOH. CONCLUSIONS: Positive charges on the surface of PAMAM dendrimer promote its exocytosis in MCF-7/ADR cells. Three intracellular transportation processes, attraction to the mitochondria and cell nucleus, as well as nuclear efflux generated by MVP led to the highest exocytosis observed for PAMAM-NH2. Our findings provide theoretical guidance to design a surface-charged tumor-targeting drug delivery system with highly efficient transfection in multidrug-resistant tumor cells. Especially, to provide more DNA to the nucleus and enhance DNA transfection efficiency in multidrug-resistant tumor cells using PAMAM-NH2, siRNA-MVP or an inhibitor should be codelivered to decrease MVP-mediated nuclear efflux.
Assuntos
Neoplasias da Mama/metabolismo , Dendrímeros/química , Exocitose/efeitos dos fármacos , Poliaminas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Dendrímeros/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células MCF-7 , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Organelas , Poliaminas/farmacologia , RNA Interferente Pequeno/metabolismo , Transfecção , Partículas de Ribonucleoproteínas em Forma de AbóbadaRESUMO
The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).
Assuntos
Acil-Butirolactonas/metabolismo , Apoptose , Bactérias/imunologia , Bactérias/metabolismo , Imunomodulação , Transdução de Sinais , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Fenômenos Fisiológicos Bacterianos , Cromatografia Líquida , Humanos , Vigilância Imunológica , Espectrometria de Massas , Proteômica/métodosRESUMO
Hedgehog signaling plays a pivotal role in embryonic pattern formation and diverse aspects of the postnatal biological process. Perturbation of the hedgehog pathway and overexpression of GLI1, a downstream transcription factor in the hedgehog pathway, are highly relevant to several malignancies including chondrosarcoma (CS). We previously found that knocking down expression of GLI1 attenuates the disrupted Indian hedgehog (IHH) signal pathway and suppresses cell survival in human CS cells. However, the underlying mechanisms regulating the expression of GLI1 are still unknown. Here, we demonstrated the implication of GLI1 in SMO-independent pathways in CS cells. A GLI1 binding protein, major vault protein (MVP), was identified using the affinity purification method. MVP promoted the nuclear transport and stabilization of GLI1 by compromising the binding affinity of GLI1 with suppressor of fused homolog (SUFU) and increased GLI1 expression via mTOR/S6K1 signaling cascade. Functionally, knockdown of MVP suppressed cell growth and induced apoptosis. Simultaneous inhibition of MVP and GLI1 strongly inhibits the growth of CS in vitro and in vivo. Moreover, IHC results showed that MVP, GLI1, and P-p70S6K1 were highly expressed and positively correlated with each other in 71 human CS tissues. Overall, our findings revealed a novel regulating mechanism for HH-independent GLI1 expression and provide a rationale for combination therapy in patients with advanced CS.
Assuntos
Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Condrossarcoma/genética , Condrossarcoma/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
The demographic shift toward an older population will increase the number of prostate cancer cases. A challenge in the treatment of prostate cancer is to avoid undertreatment of patients at high risk of progression following curative treatment. These men can benefit from early salvage treatment. An explorative cohort consisting of tissue from 16 patients who underwent radical prostatectomy, and were either alive or had died from prostate cancer within 10 years postsurgery, was analyzed by mass spectrometry analysis. Following proteomic and bioinformatic analyses, major vault protein (MVP) was identified as a putative prognostic biomarker. A publicly available tissue proteomics dataset and a retrospective cohort of 368 prostate cancer patients were used for validation. The prognostic value of the MVP was verified by scoring immunohistochemical staining of a tissue microarray. High level of MVP was associated with more than 4-fold higher risk for death from prostate cancer (hazard ratio = 4.41, 95% confidence interval: 1.45-13.38; P = 0.009) in a Cox proportional hazard models, adjusted for Cancer of the Prostate Risk Assessments Post-surgical (CAPRA-S) score and perineural invasion. Decision curve analyses suggested an improved standardized net benefit, ranging from 0.06 to 0.18, of adding MVP onto CAPRA-S score. This observation was confirmed by receiver operator characteristics curve analyses for the CAPRA-S score versus CAPRA-S and MVP score (area under the curve: 0.58 versus 0.73). From these analyses, one can infer that MVP levels in combination with CAPRA-S score might add onto established risk parameters to identify patients with lethal prostate cancer.
Assuntos
Neoplasias da Próstata/genética , Proteômica , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Biomarcadores Tumorais/genética , Evolução Fatal , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologiaRESUMO
The small noncoding vault RNA (vtRNA) is a component of the vault complex, a ribonucleoprotein complex found in most eukaryotes. Emerging evidence suggests that vtRNAs may be involved in the regulation of a variety of cellular functions when unassociated with the vault complex. Here, we demonstrate a novel role for vtRNA in synaptogenesis. Using an in vitro synapse formation model, we show that murine vtRNA (mvtRNA) promotes synapse formation by modulating the MAPK signaling pathway. mvtRNA is transported to the distal region of neurites as part of the vault complex. Interestingly, mvtRNA is released from the vault complex in the neurite by a mitotic kinase Aurora-A-dependent phosphorylation of MVP, a major protein component of the vault complex. mvtRNA binds to and activates MEK1 and thereby enhances MEK1-mediated ERK activation in neurites. These results suggest the existence of a regulatory mechanism of the MAPK signaling pathway by vtRNAs as a new molecular basis for synapse formation.
Assuntos
Sistema de Sinalização das MAP Quinases , Pequeno RNA não Traduzido/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Aurora Quinase A/metabolismo , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cinesinas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neuritos/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Densidade Pós-Sináptica/efeitos dos fármacos , Densidade Pós-Sináptica/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Sinapses/efeitos dos fármacos , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
Lipid rafts, also known as microdomains, are important components of cell membranes and are enriched in cholesterol, glycophospholipids and receptors. They are involved in various essential cellular processes, including endocytosis, exocytosis and cellular signaling. Receptors are concentrated at lipid rafts, through which cellular signaling can be transmitted. Pathogens exploit these signaling mechanisms to enter cells, proliferate and egress. However, lipid rafts also play an important role in initiating antimicrobial responses by sensing pathogens via clustered pathogen-sensing receptors and triggering downstream signaling events such as programmed cell death or cytokine production for pathogen clearance. In this review, we discuss how both host and pathogens use lipid rafts and associated proteins in an arms race to survive. Special attention is given to the involvement of the major vault protein, the main constituent of a ribonucleoprotein complex, which is enriched in lipid rafts upon infection with vaccinia virus.