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Although autophagy regulates the quality and quantity of cellular organelles, the regulatory mechanisms of peroxisomal autophagy remain largely unknown. In this study, we developed a cell-based image screening assay, and identified 1,10-phenanthroline (Phen) as a novel pexophagy inducer from chemical library screening. Treatment with Phen induces selective loss of peroxisomes but not endoplasmic reticulum and Golgi apparatus in hepatocytes. In addition, Phen increases autophagic engulfment of peroxisomes in an ATG5 dependent manner. Interestingly, treatment of Phen excessively produces peroxisomal reactive oxygen species (ROS), and inhibition of the ROS suppresses loss of peroxisome in Phen-treated cells. Taken together, these results suggest that Phen triggers pexophagy by enhancing peroxisomal ROS.
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Autofagia/efectos de los fármacos , Quelantes/farmacología , Peroxisomas/efectos de los fármacos , Fenantrolinas/farmacología , Especies Reactivas de Oxígeno/agonistas , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Catalasa/genética , Catalasa/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Peroxisomas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
The gut microbiome significantly influences immune responses and the efficacy of immune checkpoint inhibitors. We conducted a clinical trial (NCT04264975) combining an anti-programmed death-1 (PD-1) inhibitor with fecal microbiota transplantation (FMT) from anti-PD-1 responder in 13 patients with anti-PD-1-refractory advanced solid cancers. FMT induced sustained microbiota changes and clinical benefits in 6 of 13 patients, with 1 partial response and 5 stable diseases, achieving an objective response rate of 7.7% and a disease control rate of 46.2%. The clinical response correlates with increased cytotoxic T cells and immune cytokines in blood and tumors. We isolated Prevotella merdae Immunoactis from a responder to FMT, which stimulates T cell activity and suppresses tumor growth in mice by enhancing cytotoxic T cell infiltration. Additionally, we found Lactobacillus salivarius and Bacteroides plebeius may inhibit anti-tumor immunity. Our findings suggest that FMT with beneficial microbiota can overcome resistance to anti-PD-1 inhibitors in advanced solid cancers, especially gastrointestinal cancers.
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Microenvironmental acidosis is a common feature of inflammatory loci, in which clearance of apoptotic cells is necessary for the resolution of inflammation. Although it is known that a low pH environment affects immune function, its effect on apoptotic cell clearance by macrophages has not been fully investigated. Here, we show that treatment of macrophages with low pH medium resulted in increased expression of stabilin-1 out of several receptors, which are known to be involved in PS-dependent removal of apoptotic cells. Reporter assays showed that the -120/-1 region of the mouse stabilin-1 promoter was a low pH-responsive region and provided evidence that extracellular low pH mediated transcriptional activation of stabilin-1 via Ets-2. Furthermore, extracellular low pH activated JNK, thereby inducing translocation of Ets-2 into the nucleus. When macrophages were preincubated with low pH medium, phagocytosis of phosphatidylserine-exposed red blood cells and phosphatidylserine-coated beads by macrophages was enhanced. Blockade of stabilin-1 in macrophages abolished the enhancement of phagocytic activity by low pH. Thus, our results demonstrate that a low pH microenvironment up-regulates stabilin-1 expression in macrophages, thereby modulating the phagocytic capacity of macrophages, and suggest roles for stabilin-1 and Ets-2 in the maintenance of tissue homeostasis by the immune system.
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Moléculas de Adhesión Celular Neuronal/genética , Espacio Extracelular/química , Espacio Extracelular/metabolismo , Regulación de la Expresión Génica , Macrófagos Peritoneales/citología , Fagocitosis , Fosfatidilserinas/metabolismo , Animales , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , MAP Quinasa Quinasa 4/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Ratas , Activación TranscripcionalRESUMEN
This study evaluated changes in the peripheral blood immune cell population in patients with advanced thyroid cancer receiving lenvatinib treatment to confirm the immune-modulatory effect of lenvatinib. After obtaining informed consent from patients, we prospectively collected 20 ml of whole blood at 2-3 months intervals 2-4 times from each patient; peripheral blood mononuclear cells (PBMCs) were separated, and the Maxparâ¯Direct Immune Profiling Assay was performed. A total of 10 patients were enrolled, and 31 blood samples were obtained. The median age of patients was 65 years, and all patients showed durable responses to the lenvatinib treatment. When we compared the PBMC profiles between the pre-treatment, on-treatment, and off-treatment samples, the peripheral natural killer (NK) cell proportion differed significantly. The proportion of NK cells among total live cells significantly increased from 9.3 ± 4.5 (%) in the pre-treatment samples to 20.8 ± 7.9 (%) in the on-treatment samples (P = 0.009) and decreased to 13.3 ± 3.1 (%) in the off-treatment samples (P = 0.07). There was a significant increase in the peripheral NK cell population with lenvatinib treatment in advanced thyroid cancer patients. This finding confirms the immune-modulatory effect of lenvatinib.
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Antineoplásicos , Quinolinas , Neoplasias de la Tiroides , Humanos , Anciano , Leucocitos Mononucleares , Neoplasias de la Tiroides/terapia , Compuestos de Fenilurea/uso terapéutico , Quinolinas/uso terapéutico , Antineoplásicos/uso terapéuticoRESUMEN
Immune checkpoint inhibitors (ICIs) offer improved survival for patients with advanced malignant melanomas. However, only a subset of these patients exhibit an objective response rate of 10-40 % with ICIs. We aimed to ascertain the effects of RNA signatures and the spatial distribution of immune cells on the treatment outcomes of patients with malignant melanomas undergoing ICI therapy. Clinical data were retrospectively collected from ICI-treated patients with malignant melanoma; RNA expression profiles were examined via next-generation sequencing, whereas the composition, density, and spatial distribution of immune cells were determined via multiplex immunohistochemistry. Patients with poor and good responses to ICIs showed significant differences in mRNA expression profiles. Different spatial distributions of T-cells, macrophages, and NK cells as well as RNA signatures of immune-related genes were found to be closely related to therapeutic outcomes in ICI-treated patients with malignant melanomas. The spatial distributions of PD-1+ T-cells and activated M1 macrophages showed a significant correlation with favorable responses to ICIs. Our findings highlight the clinical relevance of the spatial proximity of immune cell subsets in the treatment outcomes of metastatic malignant melanoma.
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Melanoma , Receptor de Muerte Celular Programada 1 , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Macrófagos/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Receptor de Muerte Celular Programada 1/genética , ARN , ARN Mensajero , Estudios Retrospectivos , Neoplasias Cutáneas , Melanoma Cutáneo MalignoRESUMEN
Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast and gastric cancers and this causes poor clinical outcomes. Although both T-DM1 and Enhertu are approved as an HER2-targeting antibody-drug conjugate (ADC), the effects of these drugs are still not satisfactory to eradicate diverse tumors expressing HER2. To address this shortfall in HER2-targeted therapeutics, an elaborate cleavable linker is created and a novel HER2-targeting ADC composed with trastuzumab and monomethyl auristatin F, which is being investigated in a phase 1 clinical trial and is referred to as LegoChem Bisciences-ADC (LCB-ADC). LCB-ADC displays a higher cytotoxic potency than T-DM1 and it also has a higher G2/M arrest ratio. In animal studies, LCB-ADC produces noticeable tumor growth inhibition compared with trastuzumab or T-DM1 in an HER2 high-expressing N87 xenograft tumor. Especially, LCB-ADC shows good efficacy in terms of suppressing tumor growth in a patient-derived xenograft (PDX) model of HER2-positive gastric cancer as well as in T-DM1-resistant models such as HER2 low-expressing HER2 low expressing JIMT-1 xenograft tumor and PDX. Collectively, the results demonstrate that LCB-ADC with the elaborate linker has a higher efficacy and greater biostability than its ADC counterparts and may successfully treat cancers that are nonresponsive to previous therapeutics.
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Antineoplásicos Inmunológicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Oligopéptidos/uso terapéutico , Receptor ErbB-2/genética , Neoplasias Gástricas/tratamiento farmacológico , Trastuzumab/uso terapéutico , Animales , Modelos Animales de Enfermedad , Haplorrinos , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Ratas , Neoplasias Gástricas/genética , Neoplasias Gástricas/inmunologíaRESUMEN
Fucosylation is a posttranslational modification that attaches fucose residues to protein or lipidbound oligosaccharides. Certain fucosylation pathway genes are aberrantly expressed in several types of cancer, including nonsmall cell lung cancer (NSCLC), and this aberrant expression is associated with poor prognosis in patients with cancer. However, the molecular mechanism by which these fucosylation pathway genes promote tumor progression has not been wellcharacterized. The present study analyzed public microarray data obtained from NSCLC samples. Multivariate analysis revealed that altered expression of fucosylation pathway genes, including fucosyltransferase 1 (FUT1), FUT2, FUT3, FUT6, FUT8 and GDPLfucose synthase (TSTA3), correlated with poor survival in patients with NSCLC. Inhibition of FUTs by 2Fperacetylfucose (2FPAF) suppressed transforming growth factor ß (TGFß)mediated Smad3 phosphorylation and nuclear translocation in NSCLC cells. In addition, woundhealing and Transwell migration assays demonstrated that 2FPAF inhibited TGFßinduced NSCLC cell migration and invasion. Furthermore, in vivo bioluminescence imaging analysis revealed that 2FPAF attenuated the metastatic capacity of NSCLC cells. These results may help characterize the oncogenic role of fucosylation in NSCLC biology and highlight its potential for developing cancer therapeutics.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Fucosa/metabolismo , Fucosiltransferasas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Anciano , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Supervivencia sin Enfermedad , Femenino , Fucosiltransferasas/antagonistas & inhibidores , Fucosiltransferasas/metabolismo , Perfilación de la Expresión Génica , Glicosilación , Humanos , Estimación de Kaplan-Meier , Pulmón/patología , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Procesamiento Proteico-Postraduccional/genética , Tasa de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the present study, we evaluated the pharmacokinetics (PK) of trastuzumab and sought to predict human PK based on animal studies, through the use of optical imaging and a whole-body physiologically based pharmacokinetic (WB-PBPK) modeling approach. The PK study was conducted in 24 mice, where serial blood samples were withdrawn and major organs were isolated after the last blood withdrawal. The drug concentrations in blood and major organs were measured via optical imaging. The WB-PBPK model was constructed using known physiological values including the volumes of major organs and blood/lymphatic flow. The NONMEM software (version 7.3) was used to determine tissue partition coefficients. Using the WB-PBPK model, a clinical trial simulation was performed with reference to human physiological values acquired from the literature. The simulated human PK was then compared with the actual PK observed in the previous study in which healthy male subjects received 6 mg/kg trastuzumab (Herceptin®) via intravenous route. The ratio of the simulated versus observed area under the concentration-time curve was 1.02 and that of maximal concentration was 0.72. The current study describes the potential synergistic applications of WB-PBPK and optical imaging in human PK prediction based on preclinical data obtained in early-stage drug development.
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Modelos Biológicos , Trastuzumab/farmacocinética , Administración Intravenosa , Animales , Área Bajo la Curva , Ensayos Clínicos como Asunto , Simulación por Computador , Evaluación Preclínica de Medicamentos , Voluntarios Sanos , Humanos , Masculino , Ratones , Modelos Animales , Equivalencia Terapéutica , Distribución Tisular , Trastuzumab/administración & dosificaciónRESUMEN
The rapid and precise clearance of apoptotic cells (efferocytosis) involves a series of phagocytic processes through which apoptotic cells are recognized, engulfed, and degraded within phagocytes. The Rho-family GTPases critically rearrange the cytoskeleton for these phagocytic processes, but we know little about the mechanisms by which regulatory proteins control the spatiotemporal activities of the Rho-family GTPases. Here, we identify ArhGAP12 as a functional GTPase-activating protein (GAP) of Rac1 during Stabilin-2 mediated efferocytosis. ArhGAP12 constitutively forms a complex with the phosphatidylserine receptor, Stabilin-2, via direct interaction with the downstream protein, GULP, but is released from the complex when Stabilin-2 interacts with apoptotic cells. When the phagocytic cup is closed and the apoptotic cell is surrounded by the phagosomal membrane, ArhGAP12 localizes to the phagocytic cup via a specific interaction with phosphatidylinositol-4,5-bisphosphate, which is transiently biosynthesized in the phagocytic cup. Down-regulation of ArhGAP12 results in sustained Rac1 activity, arrangement of F-actin, and delayed phagosome-lysosome fusion. Our results collectively suggest that ArhGAP12 carries dual roles in Stabilin-2 mediated efferocytosis: it binds to GULP/Stabilin-2 and switches off Rac1 basal activity and switches on the Rac1 by releasing itself from the complex. In addition, the spatiotemporal membrane targeting of ArhGAP12 inactivates Rac1 in a time-specific and spatially coordinated manner to orchestrate phagosome maturation. This may shed light on how other RhoGAPs spatiotemporally inactivate Rac or Cdc42 during phagocytosis by various cells, in different circumstances.
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Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Fagocitosis/fisiología , Fagosomas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/farmacología , Línea Celular , Regulación hacia Abajo , Proteínas Activadoras de GTPasa/genética , Técnicas de Silenciamiento del Gen , Humanos , Lisosomas/metabolismo , Ratones , Fagocitos , Fagocitosis/efectos de los fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMEN
After the publication of this work errors were noticed in Fig. 3b and 4d.
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Oxidative stress is a key mediator of neuronal death in acute brain injuries, such as epilepsy, trauma, and stroke. Although it is accompanied by diverse cellular changes, increases in levels of intracellular zinc ion (Zn2+) and calcium ion (Ca2+) may play a critical causative role in oxidative neuronal death. However, the mechanistic link between Zn2+ and Ca2+ dyshomeostasis in neurons during oxidative stress is not well-understood. Here, we show that the exposure of cortical neurons to H2O2 led to a zinc-triggered calcium influx, which resulted in neuronal death. The cyclin-dependent kinase inhibitor, NU6027, inhibited H2O2-induced Ca2+ increases and subsequent cell death in cortical neurons, without affecting the early increase in Zn2+. Therefore, we attempted to identify the zinc-regulated Ca2+ pathway that was inhibited by NU6027. The expression profile in cortical neurons identified transient receptor potential cation channel 5 (TRPC5) as a candidate that is known to involve in the generation of epileptiform burst firing and epileptic neuronal death (Phelan KD et al. 2012a; Phelan KD et al. 2013b). NU6027 inhibited basal and zinc-augmented TRPC5 currents in TRPC5-overexpressing HEK293 cells. Consistently, cortical neurons from TRPC5 knockout mice were highly resistant to H2O2-induced death. Moreover, NU6027 is neuroprotective in kainate-treated epileptic rats. Our results demonstrate that TRPC5 is a novel therapeutic target against oxidative neuronal injury in prolonged seizures and that NU6027 is a potent inhibitor of TRPC5.
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Calcio/metabolismo , Neuronas/metabolismo , Neuronas/patología , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/metabolismo , Zinc/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Muerte Celular , Células HEK293 , Humanos , Peróxido de Hidrógeno/toxicidad , Ratones Endogámicos ICR , Ratones Noqueados , Neuronas/efectos de los fármacos , Compuestos Nitrosos/farmacología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Pirimidinas/farmacología , RatasRESUMEN
BACKGROUND AND STUDY AIM: To develop a molecular imaging endoscopic system that eliminates tissue autofluorescence and distinguishes multiple fluorescent markers specifically on the cancerous lesions. METHODS: Newly developed multi-spectral fluorescence endoscope device has the potential to eliminate signal interference due to autofluorescence and multiplex fluorophores in fluorescent probes. The multiplexing capability of the multi-spectral endoscope device was demonstrated in the phantom studies and multi-spectral imaging with endoscopy and macroscopy was performed to analyze fluorescence signals after administration of fluorescent probe that targets cancer in the colon. Because of the limitations in the clinical application using rigid-type small animal endoscope, we developed a flexible channel insert-type fluorescence endoscope, which was validated on the colonoscopy of dummy and porcine model. RESULTS: We measured multiple fluorescent signals simultaneously, and the fluorescence spectra were unmixed to separate the fluorescent signals of each probe, in which multiple fluorescent probes clearly revealed spectral deconvolution at the specific targeting area in the mouse colon. The positive area of fluorescence signal for each probe over the whole polyp was segmented with analyzing software, and showed distinctive patterns and significantly distinguishable values: 0.46⯱â¯0.04, 0.39⯱â¯0.08 and 0.73⯱â¯0.12 for HMRG, CET-553 and TRA-675 probes, respectively. The spectral unmixing was finally demonstrated in the dummy and porcine model, corroborating the targeted multi-spectral fluorescence imaging of colon dysplasia. CONCLUSION: The multi-spectral endoscopy system may allow endoscopists to clearly identify cancerous lesion that has different patterns of various target expression using multiple fluorescent probes.
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Tumor heterogeneity is an important concept when assessing intratumoral variety in vascular phenotypes and responses to antiangiogenic treatment. This study explored spatiotemporal heterogeneity of vascular alterations in C6 glioma mice during tumor growth and antiangiogenic treatment on serial MR examinations (days 0, 4, and 7 from initiation of vehicle or multireceptor tyrosine kinase inhibitor administration). Transvascular permeability (TP) was quantified on dynamic-contrast-enhanced MRI (DCE-MRI) using extravascular extracellular agent (Gd-DOTA); blood volume (BV) was estimated using intravascular T2 agent (SPION). With regard to region-dependent variability in vascular phenotypes, the control group demonstrated higher TP in the tumor center than in the periphery, and greater BV in the tumor periphery than in the center. This distribution pattern became more apparent with tumor growth. Antiangiogenic treatment effect was regionally heterogeneous: in the tumor center, treatment significantly suppressed the increase in TP and decrease in BV (ie, typical temporal change in the control group); in the tumor periphery, treatment-induced vascular alterations were insignificant and BV remained high. On histopathological examination, the control group showed greater CD31, VEGFR2, Ki67, and NG2 expression in the tumor periphery than in the center. After treatment, CD31 and Ki67 expression was significantly suppressed only in the tumor center, whereas VEGFR2 and α-caspase 3 expression was decreased and NG2 expression was increased in the entire tumor. These results demonstrate that MRI can reliably depict spatial heterogeneity in tumor vascular phenotypes and antiangiogenic treatment effects. Preserved angiogenic activity (high BV on MRI and high CD31) and proliferation (high Ki67) in the tumor periphery after treatment may provide insights into the mechanism of tumor resistance to antiangiogenic treatment.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Neoplasias/patología , Neovascularización Patológica , Animales , Biomarcadores , Volumen Sanguíneo , Permeabilidad Capilar , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Ratones , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neovascularización Patológica/diagnóstico por imagen , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The removal of unwanted or damaged cells by phagocytes is achieved via a finely regulated cleaning process called efferocytosis. To characterize the mechanisms through which phagocytes control the intake of apoptotic cells, we investigated how the phagocyte's appetite for engulfed cells may be coordinated by RhoA and Rac1 in the phagocytic cup. We used FRET biosensors to visualize the spatiotemporal dynamics of Rho-family GTPases, and found that RhoA, which is known to be downregulated during phagocytosis, was transiently upregulated at the phagocytic cup immediately prior to ingestion. Conversely, Rac1 was upregulated during the engulfment process and then downregulated prior to phagosomal maturation. Moreover, disturbance of the dynamic activities of RhoA led to uncontrolled engulfment, such as fast and undiscerning eating. Our results reveal that the temporal activity of RhoA GTPase alters the Rac1/RhoA balance at the phagocytic cup prior to ingestion, and that this plays a distinct role in orchestrating efferocytosis, with RhoA modulating the rate of engulfment to ensure that the phagocyte engulfs an appropriate amount of the correct material.
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Fagocitos/fisiología , Fagocitosis/fisiología , Proteína de Unión al GTP rac1/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Apoptosis/fisiología , Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Humanos , Células L , Macrófagos Peritoneales/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Imagen de Lapso de TiempoRESUMEN
Rab escort protein 1 (REP1) is a component of Rab geranyl-geranyl transferase 2 complex. Mutations in REP1 cause a disease called choroideremia (CHM), which is an X-linked eye disease. Although it is postulated that REP1 has functions in cell survival or death of various tissues in addition to the eye, how REP1 functions in normal and cancer cells remains to be elucidated. Here, we demonstrated that REP1 is required for the survival of intestinal cells in addition to eyes or a variety of cells in zebrafish, and also has important roles in tumorigenesis. Notably, REP1 is highly expressed in colon cancer tissues and cell lines, and silencing of REP1 sensitizes colon cancer cells to serum starvation- and 5-FU-induced apoptosis. In an effort to elucidate the molecular mechanisms underlying REP1-mediated cell survival under those stress conditions, we identified FOXO3 as a binding partner of REP1 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that REP1 blocked the nuclear trans-localization of FOXO3 through physically interacting with FOXO3, thereby suppressing FOXO3-mediated apoptosis. Importantly, the inhibition of REP1 combined with 5-FU treatment could lead to significant retarded tumor growth in a xenograft tumor model of human cancer cells. Thus, our results suggest that REP1 could be a new therapeutic target in combination treatment for colon cancer patients.
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Proteínas Adaptadoras Transductoras de Señales/genética , Carcinogénesis/genética , Neoplasias del Colon/genética , Proteína Forkhead Box O3/genética , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Coroideremia/genética , Coroideremia/patología , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Fluorouracilo/administración & dosificación , Proteína Forkhead Box O3/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mutación , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genéticaRESUMEN
Interstitial cystitis/bladder pain syndrome (IC/BPS) is an intractable disease characterized by severe pelvic pain and urinary frequency. Mesenchymal stem cell (MSC) therapy is a promising approach to treat incurable IC/BPS. Here, we show greater therapeutic efficacy of human embryonic stem cell (hESC)-derived multipotent stem cells (M-MSCs) than adult bone-marrow (BM)-derived counterparts for treating IC/BPS and also monitor long-term safety and in vivo properties of transplanted M-MSCs in living animals. Controlled hESC differentiation and isolation procedures resulted in pure M-MSCs displaying typical MSC behavior. In a hydrochloric-acid instillation-induced IC/BPS animal model, a single local injection of M-MSCs ameliorated bladder symptoms of IC/BPS with superior efficacy compared to BM-derived MSCs in ameliorating bladder voiding function and histological injuries including urothelium denudation, mast-cell infiltration, tissue fibrosis, apoptosis, and visceral hypersensitivity. Little adverse outcomes such as abnormal growth, tumorigenesis, or immune-mediated transplant rejection were observed over 12-months post-injection. Intravital confocal fluorescence imaging tracked the persistence of the transplanted cells over 6-months in living animals. The infused M-MSCs differentiated into multiple cell types and gradually integrated into vascular-like structures. The present study provides the first evidence for improved therapeutic efficacy, long-term safety, and in vivo distribution and cellular properties of hESC derivatives in preclinical models of IC/BPS.
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Cistitis Intersticial/metabolismo , Cistitis Intersticial/fisiopatología , Células Madre Embrionarias Humanas/metabolismo , Trasplante de Células Madre Mesenquimatosas , Manejo del Dolor , Dolor/metabolismo , Animales , Biomarcadores , Cistitis Intersticial/etiología , Cistitis Intersticial/terapia , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Células Madre Embrionarias Humanas/citología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cariotipo , Microscopía Confocal , Imagen Molecular , Dolor/etiología , Dolor/fisiopatología , Transducción de Señal , Síndrome , Resultado del Tratamiento , Proteínas Wnt/metabolismoRESUMEN
A mitochondria-targeting ratiometric probe was designed for γ-glutamyltranspeptidase (γGT). Mechanistic study by HPLC and an inhibitor assay showed that the probe underwent γGT-mediated amide-to-amine transformation and induced a ratiometric fluorescence response in cellular mitochondria. Further application was successful for the detection of cancerous colons in mice.
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Neoplasias del Colon/diagnóstico por imagen , Colorantes Fluorescentes/química , Glutamina/análogos & derivados , Glutamina/química , Indoles/química , Mitocondrias/metabolismo , gamma-Glutamiltransferasa/análisis , Animales , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Glutamina/síntesis química , Glutamina/metabolismo , Células HCT116 , Humanos , Hidrólisis , Indoles/síntesis química , Indoles/metabolismo , Límite de Detección , Ratones , gamma-Glutamiltransferasa/metabolismoRESUMEN
Autophagy is a catabolic degradation process and maintains cellular homeostasis. And autophagy is activated in response to various stress conditions. Although O-GlcNAcylation functions a sensor for nutrient and stress, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we identified that ATG4B is novel target for O-GlcNAcylation under metabolic stress condition. Treatment with PugNAc, an O-GlcNAcase inhibitor increased activation of autophagy in SH-SY5Y cells. Both bimolecular fluorescence complementation and immunoprecipitation assay indicated that OGT directly interacts with ATG4B in SH-SY5Y cells. We also found that the O-GlcNAcylated ATG4B was increased in autophagy activation conditions, and down-regulation of OGT reduces O-GlcNAcylation of ATG4B under low glucose condition. Furthermore, the proteolytic activity of ATG4B for LC3 cleavage was enhanced in PugNAc-treated cells. Taken together, these results imply that O-GlcNAcylation of ATG4B regulates autophagy activation by increasing its proteolytic activity under metabolic stress condition.
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Proteínas Relacionadas con la Autofagia/química , Autofagia , Cisteína Endopeptidasas/química , Regulación Enzimológica de la Expresión Génica , Oxigenasas de Función Mixta/química , N-Acetilglucosaminiltransferasas/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Animales , Línea Celular Tumoral , Regulación hacia Abajo , Fibroblastos/metabolismo , Colorantes Fluorescentes/química , Glucosa/química , Humanos , Inmunoprecipitación , Luciferasas/metabolismo , Espectrometría de Masas , Ratones , Oximas/metabolismo , Fenilcarbamatos/metabolismo , Transducción de Señal , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Primary cilia have critical roles in coordinating multiple cellular signaling pathways. Dysregulation of primary cilia is implicated in various ciliopathies. To identify specific regulators of autophagy, we screened chemical libraries and identified mefloquine, an anti-malaria medicine, as a potent regulator of primary cilia in human retinal pigmented epithelial (RPE) cells. Not only ciliated cells but also primary cilium length was increased in mefloquine-treated RPE cells. Treatment with mefloquine strongly induced the elongation of primary cilia by blocking disassembly of primary cilium. In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux. Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells. Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis fruit extract (SCE) has been used as a traditional oriental medicine for treating vascular diseases. However, the pharmacologic effects and mechanisms of SCE on vascular fibrosis are still largely unknown. Transforming growth factor ß1 (TGFß1)-mediated cellular changes are closely associated with the pathogenesis of vascular fibrotic diseases. Particularly, TGFß1 induces actin stress fiber formation that is a crucial mechanism underlying vascular smooth muscle cell (VSMC) migration in response to vascular injury. In this study, we investigated the effect of SCE and its active ingredients on TGFß1-induced stress fiber assembly in A7r5 VSMCs. MATERIALS AND METHODS: To investigate pharmacological actions of SCE and its ingredients on TGFß1-treated VSMCs, we have employed molecular and cell biological technologies, such as confocal microscopy, fluorescence resonance energy transfer, western blotting, and radiometric enzyme analyses. RESULTS: We found that SCE inhibited TGFß1-induced stress fiber formation and cell migration. Schisandrin B (SchB) showed the most prominent effect among the active ingredients of SCE tested. SchB reduced TGFß1-mediated phosphorylation of myosin light chain, and this effect was independent of RhoA/Rho-associated kinase pathway. Fluorescence resonance energy transfer and radiometric enzyme assays confirmed that SchB inhibited myosin light chain kinase activity. We also showed that SchB decreased TGFß1-mediated induction of α-smooth muscle actin by inhibiting Smad signaling. CONCLUSIONS: The present study demonstrates that SCE and its active ingredient SchB suppressed TGFß1-induced stress fiber formation at the molecular level. Therefore, our findings may help future investigations to develop multi-targeted therapeutic strategies that attenuate VSMC migration and vascular fibrosis.