RESUMO
Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (|ρ| â¼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics.
RESUMO
Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune-modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical-grade NK-EVs for pre-clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed-loop hollow-fibre bioreactor to continuously produce NK-EVs from the clinically relevant NK92-MI cell line under serum-free, Xeno-free and feeder-free conditions following GMP-compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK-EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro-inflammatory cytokine (interferon-gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off-target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell-based and NK-EV-based immunotherapies for cancer with GMP considerations.
Assuntos
Vesículas Extracelulares , Neoplasias , Humanos , Fluxo de Trabalho , Células Matadoras Naturais , Imunoterapia/métodos , Neoplasias/terapia , Neoplasias/metabolismoRESUMO
Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry.IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.
Assuntos
Ebolavirus/metabolismo , Proteína C1 de Niemann-Pick/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Ebolavirus/genética , Ebolavirus/patogenicidade , Endossomos/metabolismo , Filoviridae/genética , Infecções por Filoviridae/genética , Infecções por Filoviridae/metabolismo , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/metabolismo , Interações Hospedeiro-Patógeno , Glicoproteínas de Membrana/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Receptores Virais/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
Filoviruses, such as Ebola virus (EBOV) and Marburg virus, are causative agents of unpredictable outbreaks of severe hemorrhagic fevers in humans and non-human primates. For infection, filoviral particles need to be internalized and delivered to intracellular vesicles containing cathepsin proteases and the viral receptor Niemann-Pick C1. Previous studies have shown that EBOV triggers macropinocytosis of the viral particles in a glycoprotein (GP)-dependent manner, but the molecular events required for filovirus internalization remain mostly unknown. Here we report that the diacylglycerol kinase inhibitor, R-59-022, blocks EBOV GP-mediated entry into Vero cells and bone marrow-derived macrophages. Investigation of the mode of action of the inhibitor revealed that it blocked an early step in entry, more specifically, the internalization of the viral particles via macropinocytosis. Finally, R-59-022 blocked viral entry mediated by a panel of pathogenic filovirus GPs and inhibited growth of replicative Ebola virus. Taken together, our studies suggest that R-59-022 could be used as a tool to investigate macropinocytic uptake of filoviruses and could be a starting point for the development of pan-filoviral therapeutics.
Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Filoviridae/efeitos dos fármacos , Filoviridae/fisiologia , Pirimidinonas/farmacologia , Tiazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Chlorocebus aethiops , Ebolavirus/fisiologia , Células HEK293 , Humanos , Macrófagos/virologia , Marburgvirus/fisiologia , Pinocitose/efeitos dos fármacos , Receptores Virais , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P2-positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P2 production in cells to promote efficient delivery to NPC1.
Assuntos
Proteínas de Transporte/metabolismo , Ebolavirus/fisiologia , Glicoproteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Flavoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Proteína C1 de Niemann-Pick , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Primary tumors are often heterogeneous, composed of therapy-sensitive and emerging therapy-resistant cancer cells. Interestingly, treatment of therapy-sensitive tumors in heterogeneous tumor microenvironments results in apoptosis of therapy-resistant tumors. In this study, we identify a prostate apoptosis response-4 (Par-4) amino-terminal fragment (PAF) that is released by diverse therapy-sensitive cancer cells following therapy-induced caspase cleavage of the tumor suppressor Par-4 protein. PAF caused apoptosis in cancer cells resistant to therapy and inhibited tumor growth. A VASA segment of Par-4 mediated its binding and degradation by the ubiquitin ligase Fbxo45, resulting in loss of Par-4 proapoptotic function. Conversely, PAF, which contains this VASA segment, competitively bound to Fbxo45 and rescued Par-4-mediated induction of cancer cell-specific apoptosis. Collectively, our findings identify a molecular decoy naturally generated during apoptosis that inhibits a ubiquitin ligase to overcome therapy resistance in tumors. Cancer Res; 77(15); 4039-50. ©2017 AACR.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Experimentais/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Nus , Peptídeos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The guardian of the genome, p53, is often mutated in cancer and may contribute to therapeutic resistance. Given that p53 is intact and functional in normal tissues, we harnessed its potential to inhibit the growth of p53-deficient cancer cells. Specific activation of p53 in normal fibroblasts selectively induced apoptosis in p53-deficient cancer cells. This paracrine effect was mediated by p53-dependent secretion of the tumor suppressor Par-4. Accordingly, the activation of p53 in normal mice, but not p53(-)/(-) or Par-4(-)/(-) mice, caused systemic elevation of Par-4, which induced apoptosis of p53-deficient tumor cells. Mechanistically, p53 induced Par-4 secretion by suppressing the expression of its binding partner, UACA, which sequesters Par-4. Thus, normal cells can be empowered by p53 activation to induce Par-4 secretion for the inhibition of therapy-resistant tumors.
Assuntos
Apoptose , Comunicação Parácrina , Receptores de Trombina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Autoantígenos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Trombina/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Tumor suppressor PAR-4 acts in part by modulating sensitivity to apoptosis, but the basis for its activity is not fully understood. In this study, we describe a novel mechanism of antiapoptosis by NF-κB, revealing that it can block PAR-4-mediated apoptosis by downregulating trafficking of the PAR-4 receptor GRP78 from the endoplasmic reticulum to the cell surface. Mechanistic investigations revealed that NF-κB mediated this antiapoptotic mechanism by upregulating expression of UACA, a proinflammatory protein in certain disease settings. In clinical specimens of cancer, a strong correlation existed between NF-κB activity and UACA expression, relative to normal tissues. UACA bound to intracellular PAR-4 in diverse cancer cells, where it prevented translocation of GRP78 from the endoplasmic reticulum to the cell surface. This pathway of antiapoptosis could be inhibited by suppressing levels of NF-κB or UACA expression, which enhanced endoplasmic reticulum stress and restored GRP78 trafficking to the cell surface, thereby sensitizing cancer cells to apoptosis by extracellular PAR-4 or GRP78 agonistic antibody. In summary, our results identify a novel intracellular pathway of apoptosis mediated by NF-κB through UACA elevation, which by attenuating endoplasmic reticulum stress and GRP78 translocation to the cell surface can blunt the sensitivity of cancer cells to apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Sobrevivência Celular , NF-kappa B/metabolismo , Neoplasias/metabolismo , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Espaço Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Transporte Proteico , Receptores Ativados por Proteinase/metabolismo , Transdução de SinaisRESUMO
The tumor suppressor protein Par-4 (Prostate apoptosis response-4) is spontaneously secreted by normal and cancer cells. Extracellular Par-4 induces caspase-dependent apoptosis in cancer cell cultures by binding, via its effector SAC domain, to cell surface GRP78 receptor. However, the functional significance of extracellular Par-4/SAC has not been validated in animal models. We show that Par-4/SAC-transgenic mice express systemic Par-4/SAC protein and are resistant to the growth of non-autochthonous tumors. Consistently, secretory Par-4/SAC pro-apoptotic activity can be transferred from these cancer-resistant transgenic mice to cancer-susceptible mice by bone marrow transplantation. Moreover, intravenous injection of recombinant Par-4 or SAC protein inhibits metastasis of cancer cells. Collectively, our findings indicate that extracellular Par-4/SAC is systemically functional in inhibition of tumor growth and metastasis progression, and may merit investigation as a therapy.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Neoplasias/patologia , Proteínas Supressoras de Tumor/metabolismo , Células 3T3 , Animais , Proteínas Reguladoras de Apoptose/administração & dosagem , Proteínas Reguladoras de Apoptose/genética , Transplante de Medula Óssea , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Genes Supressores de Tumor , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/administração & dosagem , Transplante Heterólogo , Proteínas Supressoras de Tumor/genéticaRESUMO
Prostate apoptosis response-4 (Par-4) is a proapoptotic protein with intracellular functions in the cytoplasm and nucleus. Unexpectedly, we noted Par-4 protein is spontaneously secreted by normal and cancer cells in culture, and by Par-4 transgenic mice that are resistant to spontaneous tumors. Short exposure to endoplasmic reticulum (ER) stress-inducing agents further increased cellular secretion of Par-4 by a brefeldin A-sensitive pathway. Secretion occurred independently of caspase activation and apoptosis. Interestingly, extracellular Par-4 induced apoptosis by binding to the stress response protein, glucose-regulated protein-78 (GRP78), expressed at the surface of cancer cells. The interaction of extracellular Par-4 and cell surface GRP78 led to apoptosis via ER stress and activation of the FADD/caspase-8/caspase-3 pathway. Moreover, apoptosis inducible by TRAIL, which also exerts cancer cell-specific effects, is dependent on extracellular Par-4 signaling via cell surface GRP78. Thus, Par-4 activates an extrinsic pathway involving cell surface GRP78 receptor for induction of apoptosis.
Assuntos
Apoptose , Receptores de Trombina/metabolismo , Animais , Brefeldina A/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Receptores de Trombina/químicaRESUMO
The regulation of DNA relaxation by topoisomerase 1 (TOP1) is essential for DNA replication, transcription, and recombination events. TOP1 activity is elevated in cancer cells, yet the regulatory mechanism restraining its activity is not understood. We present evidence that the tumor suppressor protein prostate apoptosis response-4 (Par-4) directly binds to TOP1 and attenuates its DNA relaxation activity. Unlike camptothecin, which binds at the TOP1-DNA interface to form cleavage complexes, Par-4 interacts with TOP1 via its leucine zipper domain and sequesters TOP1 from the DNA. Par-4 knockdown by RNA interference enhances DNA relaxation and gene transcription activities and promotes cellular transformation in a TOP1-dependent manner. Conversely, attenuation of TOP1 activity either by RNA interference or Par-4 overexpression impedes DNA relaxation, cell cycle progression, and gene transcription activities and inhibits transformation. Collectively, our findings suggest that Par-4 serves as an intracellular repressor of TOP1 catalytic activity and regulates DNA topology to suppress cellular transformation.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Receptores de Trombina/metabolismo , Animais , Sequência de Bases , Linhagem Celular Transformada , DNA/química , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Imuno-Histoquímica , Imunoprecipitação , Zíper de Leucina , Camundongos , Células NIH 3T3 , Ligação ProteicaRESUMO
Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal/immortalized cells. The cancer-specific proapoptotic action of Par-4 is encoded in its centrally located SAC domain. We report here the characterization of a novel mouse model with ubiquitous expression of the SAC domain. Although SAC transgenic mice displayed normal development and life span, they were resistant to the growth of spontaneous, as well as oncogene-induced, autochthonous tumors. Resistance to tumorigenesis was linked to inhibition of nuclear factor-kappaB activity and induction of apoptosis by the SAC domain. Collectively, our findings provide genetic evidence that the SAC domain of Par-4 confers cancer resistance in transgenic mice without compromising normal viability or aging, and may have therapeutic significance.