RESUMO
Hepatic fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM), primarily collagen, within the liver. Because reactive oxygen species (ROS) has been implicated in its pathogenesis, the use of antioxidants as a potential treatment has been broadly explored. Here, we investigated the hepatoprotective properties of ramalin (RM), a compound extracted from the Antarctic lichen Ramalina terebrata, against hepatic fibrosis in vitro and in vivo. RM suppressed hepatic stellate cell (HSC) activation in vitro without any significant signs of adverse effects on the cells tested, and the accumulation of ECM was dramatically reduced in the liver tissue. Oral administration of RM in rats noticeably improved the gross appearance of the liver with increased body and liver weight relative to the DMN injected rats, and all of the serum biochemical markers returned to the normal range. RM treatment have ameliorated hepatic fibrosis in rats induced by DMN by repressing α-smooth muscle actin (α-SMA) and upregulating heme oxygenase-1 (HO-1). In addition, RM significantly reduced collagen accumulation, and levels of malondialdehyde (MDA) and hydroxyproline (HP) in the liver tissue of DMN injected rats. The efficacy exerted by RM was through erythroid 2-related factor 2 (Nrf2) mediated antioxidant response proteins such as HO-1 and NAD(P)H quinone dehydrogenase 1 (NQO-1). Our results show the beneficial effect of RM against the progression of hepatic fibrosis.
Assuntos
Antioxidantes/uso terapêutico , Glutamatos/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Animais , Antioxidantes/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dimetilnitrosamina , Progressão da Doença , Glutamatos/química , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Ratos , Elementos de Resposta , Transdução de Sinais/efeitos dos fármacosRESUMO
Mitochondria-specific delivery methods offer a valuable tool for studying mitochondria-related diseases and provide breakthroughs in therapeutic development. Although several small-molecule and peptide-based transporters have been developed, peptoids, proteolysis-resistant peptidomimetics, are a promising alternative to current approaches. We designed a series of amphipathic peptoids and evaluated their cellular uptake and mitochondrial localization. Two peptoids with cyclohexyl residues demonstrated highly efficient cell penetration and mitochondrial localization without significant adverse effects on the cells and mitochondria. These mitochondria-targeting peptoids could facilitate the selective and robust targeted delivery of bioactive compounds, such as drugs, antioxidants, and photosensitizers, with minimal off-target effects.
Assuntos
Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Mitocôndrias/metabolismo , Peptoides/metabolismo , Linhagem Celular , Portadores de Fármacos/efeitos adversos , Portadores de Fármacos/análise , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/metabolismo , Peptoides/efeitos adversos , Peptoides/análiseRESUMO
Myeloid-derived suppressor cells (MDSCs) regulate T cell immunity, and this population is a new therapeutic target for immune regulation. A previous study showed that transforming growth factor-ß (TGF-ß) is involved in controlling MDSC differentiation and immunoregulatory function in vivo. However, the direct effect of TGF-ß on MDSCs with various cytokines has not previously been tested. Thus, we examined the effect of various cytokine combinations with TGF-ß on MDSCs derived from bone marrow cells. The data show that different cytokine combinations affect the differentiation and immunosuppressive functions of MDSCs in different ways. In the presence of TGF-ß, interleukin-6 (IL-6) was the most potent enhancer of MDSC function, whereas granulocyte colony-stimulating factors (G-CSF) was the most potent in the absence of TGF-ß. In addition, IL-4 maintained MDSCs in an immature state with an increased expression of arginase 1 (Arg1). However, regardless of the cytokine combinations, TGF-ß increased expansion of the monocytic MDSC (Mo-MDSC) population, expression of immunosuppressive molecules by MDSCs, and the ability of MDSCs to suppress CD4⺠T cell proliferation. Thus, although different cytokine combinations affected the MDSCs in different ways, TGF-ß directly affects monocytic-MDSCs (Mo-MDSCs) expansion and MDSCs functions.
Assuntos
Diferenciação Celular , Interleucinas/metabolismo , Células Supressoras Mieloides/imunologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Feminino , Interleucinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/citologia , Fator de Crescimento Transformador beta/genéticaRESUMO
By utilizing a multimodal nonlinear optical system that combines coherent anti-Stokes Raman scattering and second harmonic generation to investigate biological characteristics of dermal tissues ex vivo, we demonstrate the potential feasibility of using this optical approach as a powerful new investigative tool for future biomedical research. For this study, our optical system was utilized for the first time to analyze lipid and collagen profiles in cereblon knockout (KO) mouse skin, and we were able to discover significant alterations in the number of carbon-carbon double bonds (wild-type vs. cereblon KO; NCC : 0.75 vs. 0.85) of skin fatty acids in triacylglycerides as well as changes in dermal collagen fibers (25% reduction in cereblon KO). By adopting our optical system to biological studies, we provide researchers with another diagnostic approach to validate their experimental results, which will significantly advance the state of biomedical research.
Assuntos
Colágeno , Dispositivos Ópticos , Camundongos , Animais , Pele , Ácidos Graxos , Análise Espectral RamanRESUMO
Adverse effects of spaceflight on the human body are attritubuted to microgravity and space radiation. One of the most sensitive organs affected by them is the eye, particularly the retina. The conditions that astronauts suffer, such as visual acuity, is collectively called a spaceflight-associated neuro-ocular syndrome (SANS); however, the underlying molecular mechanism of the microgravity-induced ocular pathogenesis is not clearly understood. The current study explored how microgravity affects the retina function in ARPE19 cells in vitro under time-averaged simulated microgravity (µG) generated by clinostat. We found multicellular spheroid (MCS) formation and a significantly decreased cell migration potency under µG conditions compared to 1G in ARPE19 cells. We also observed that µG increases intracellular reactive oxygen species (ROS) and causes mitochondrial dysfunction in ARPE19 cells. Subsequently, we showed that µG activates autophagic pathways and ciliogenesis. Furthermore, we demonstrated that mitophagy activation is triggered via the mTOR-ULK1-BNIP3 signaling axis. Finally, we validated the effectiveness of TPP-Niacin in mitigating µG-induced oxidative stress and mitochondrial dysfunction in vitro, which provides the first experimental evidence for TPP-Niacin as a potential therapeutic agent to ameliorate the cellular phenotypes caused by µG in ARPE19 cells. Further investigations are, however, required to determine its physiological functions and biological efficacies in primary human retinal cells, in vivo models, and target identification.
Assuntos
Niacina , Ausência de Peso , Humanos , Niacina/metabolismo , Niacina/farmacologia , Estresse Oxidativo , Células Epiteliais/metabolismo , Retina/metabolismo , Mitocôndrias/metabolismoRESUMO
It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-(13) C(2) ]glucose. The [3-(13) C]lactate/[2,3-(13) C(2) ]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, (13) C-(13) C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, (13) C multiplets of γ-aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that (13) C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy.
Assuntos
Neoplasias Encefálicas/metabolismo , Ciclo do Ácido Cítrico , Glucose/metabolismo , Glicólise , Via de Pentose Fosfato , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/secundário , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/patologia , Modelos Animais de Doenças , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Neoplasias Renais/enzimologia , Neoplasias Renais/patologia , Ácido Láctico/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Tomografia por Emissão de Pósitrons , Ácido gama-Aminobutírico/metabolismoRESUMO
In this study, we designed photo-triggered reactive oxygen species (ROS)-generating pheophorbide A and ROS-cleavable thioketal-SN38 conjugated hyaluronan-cholesterol nanoparticles (PheoA-SN38-HC NPs). And we observed the combined therapeutic effects of PheoA-SN38-HC NPs against HEY-T30 human ovarian cancer (OC) model. Clinical Proteomic Tumor Analysis Consortium (CPTAC) data showed that the expression of cancer stem cell (CSC) markers (CD44, ALDH1A1, and CD117) is highly associated with poor clinical outcomes in OC patients. We proved that HEY-T30 cells overexpress CSC markers and much more invasive than other cancer cells. Flow cytometry (FACS) and microscopic analysis revealed the active targeting property of PheoA-SN38-HC NPs to CD44+ HEY-T30 cells. Moreover, the combination therapeutic effect of PheoA-SN38-HC NPs was clearly demonstrated against in vitro HEY-T30 cells and an in vivo xenograft mouse model. In particular, the paracrine cytotoxic effect of SN38 probably compensates the locoregional therapeutic limitation of photodynamic therapy.
Assuntos
Nanopartículas , Neoplasias Ovarianas , Animais , Linhagem Celular Tumoral , Clorofila/análogos & derivados , Feminino , Humanos , Ácido Hialurônico , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Proteômica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cereblon (CRBN) is a substrate receptor of the cullin-RING E3 ubiquitin ligase (CRL) complex that mediates the ubiquitination of several substrates. In this study, CRBN knockout (KO) mice exhibited decreased levels of stratum corneum hydration (SCH) and collagen I expression with an elevated protein level of matrix metalloprotease 1 (MMP1). The absence of cereblon in the skin of CRBN KO mice mimics the damage caused by narrowband ultraviolet B (NB-UVB). The primary CRBN deficient mouse embryonic fibroblasts (MEFs) undergo G2/M-arrested premature senescence via protein signaling of p38 MAPK and its dependent p53/p21pathway. The absence of CRBN induced the markers of cellular senescence, such as the senescence-associated heterochromatin foci (SAHF), SA-ß-Gal staining, and p21 upregulation while the ectopic expression of CRBN reversed the phenotypes of SA-ß-Gal staining and p21 upregulation. Reversion of the decreased protein level of collagen I was demonstrated after the reintroduction of the CRBN gene back into CRBN KO MEFs, validating the promising role of CRBN as a potential regulator for the function of the skin barrier and its cellular homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Colágeno Tipo I/metabolismo , Sistema de Sinalização das MAP Quinases , Pele/metabolismo , Envelhecimento/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/fisiologia , Imunofluorescência , Hylobatidae , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Pele/patologia , Fenômenos Fisiológicos da PeleRESUMO
Calcium flux regulating intracellular calcium levels is essential and modulated for efficient efferocytosis. However, the molecular mechanism by which calcium flux is modulated during efferocytosis remains elusive. Here, we report that Orai1, a Crbn substrate, is upregulated via its attenuated interaction with Crbn during efferocytosis, which increases calcium influx into phagocytes and thereby promotes efferocytosis. We found that Crbn deficiency promoted phagocytosis of apoptotic cells, which resulted from facilitated phagocytic cup closure and was nullified by a CRAC channel inhibitor. In addition, Orai1 associated with Crbn, resulting in ubiquitination and proteasomal degradation of Orai1 and alteration of SOCE-mediated calcium influx. The association of Orai1 with Crbn was attenuated during efferocytosis, leading to reduced ubiquitination of Orai1 and consequently upregulation of Orai1 and calcium influx. Collectively, our study reveals a regulatory mechanism by which calcium influx is modulated by a Crbn-Orai1 axis to facilitate efferocytosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cálcio/metabolismo , Proteína ORAI1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Morte Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Ubiquitina-Proteína Ligases/genéticaRESUMO
Background: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers with a poor prognosis. Several commonly investigated immunohistochemical markers in resected HCC have potential prognostic value, but the prognostic utility of p53 expression in HCC has remained elusive. Aim: To evaluate the prognostic value of p53 and p53 phosphorylation at serine 15 (p53 Ser15-P) in patients with HCC. Methods: Surgically resected tumors from 199 HCC patients were analyzed for p21, p53, p53 Ser15-P, and proliferating cell nuclear antigen (PCNA) expression using immunohistochemistry. Results: Stratifying by the expression of p53 Ser15-P (P = 0.016), but not by p53 (P = 0.301), revealed significantly different survival outcomes in patients with HCC. Moreover, our analysis demonstrated that patients who were PCNA-positive and p53 Ser15-P-negative had significantly worse survival outcomes (P = 0.001) than patients who were PCNA-positive and p53 Ser15-P-positive. Conclusions: P53 Ser15-P is associated with poor outcomes in patients with HCC, and this prognostic marker is useful for predicting the survival of patients with PCNA-positive HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Taxa de SobrevidaRESUMO
Screening a compound library of quinolinone derivatives identified compound 11a as a new P2X7 receptor antagonist. To optimize its activity, we assessed structure-activity relationships (SAR) at three different positions, R1, R2 and R3, of the quinolinone scaffold. SAR analysis suggested that a carboxylic acid ethyl ester group at the R1 position, an adamantyl carboxamide group at R2 and a 4-methoxy substitution at the R3 position are the best substituents for the antagonism of P2X7R activity. However, because most of the quinolinone derivatives showed low inhibitory effects in an IL-1ß ELISA assay, the core structure was further modified to a quinoline skeleton with chloride or substituted phenyl groups. The optimized antagonists with the quinoline scaffold included 2-chloro-5-adamantyl-quinoline derivative (16c) and 2-(4-hydroxymethylphenyl)-5-adamantyl-quinoline derivative (17k), with IC50 values of 4 and 3â¯nM, respectively. In contrast to the quinolinone derivatives, the antagonistic effects of the quinoline compounds (16c and 17k) were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1ß, from LPS/IFN-γ/BzATP-stimulated THP-1â¯cells (IC50 of 7 and 12â¯nM, respectively). In addition, potent P2X7R antagonists significantly inhibited the sphere size of TS15-88 glioblastoma cells.
Assuntos
Antineoplásicos/farmacologia , Glioblastoma/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/farmacologia , Quinolinas/farmacologia , Quinolonas/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Humanos , Antagonistas do Receptor Purinérgico P2X/síntese química , Antagonistas do Receptor Purinérgico P2X/química , Quinolinas/síntese química , Quinolinas/química , Quinolonas/síntese química , Quinolonas/química , Receptores Purinérgicos P2X7/metabolismo , Relação Estrutura-AtividadeRESUMO
PDK1 is essential for T cell receptor (TCR)-mediated activation of NF-κB, and PDK1-induced phosphorylation of PKCθ is important for TCR-induced NF-κB activation. However, inverse regulation of PDK1 by PKCθ during T cell activation has not been investigated. In this study, we found that PKCθ is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type PKCθ or of kinase-inactive form of PKCθ revealed that PKCθ induced phosphorylation of human PDK1 at Ser-64. This PKCθ-induced PDK1 phosphorylation positively regulated T cell activation and TCR-induced NF-κB activation. Moreover, phosphorylation of human PDK1 at Ser-64 increased the stability of human PDK1 protein. These results suggest that Ser-64 is an important phosphorylation site that is part of a positive feedback loop for human PDK1-PKCθ-mediated T cell activation.
Assuntos
Isoenzimas/imunologia , Proteína Quinase C/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Estabilidade Enzimática , Células HEK293 , Humanos , Interleucina-2/biossíntese , Isoenzimas/metabolismo , Ativação Linfocitária , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Serina/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2) transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc-finger E-box binding homeobox 1 (ZEB1), which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Fator de Transcrição 2 de Oligodendrócitos/genética , Fatores de Transcrição SOXB1/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Gradação de Tumores , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Plicamicina/farmacologia , Quinazolinas/uso terapêutico , Interferência de RNA , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição SOXB1/antagonistas & inibidores , Fatores de Transcrição SOXB1/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
In this study, we prepared macromolecular MR T1 contrast agent: pullulan-conjugated Gd diethylene triamine pentaacetate (Gd-DTPA-Pullulan) and estimated residual free Gd(3+), chelation stability in competition with metal ions, plasma and tissue pharmacokinetics, and abdominal MR contrast on rats. Residual free Gd(3+) in Gd-DTPA-Pullulan was measured using colorimetric spectroscopy. The transmetalation of Gd(3+) incubated with Ca(2+) was performed by using a dialysis membrane (MWCO 100-500 Da) and investigated by ICP-OES. The plasma concentration profiles of Gd-DTPA-Pullulan were estimated after intravenous injection at a dose 0.1 mmol/kg of Gd. The coronal-plane abdominal images of normal rats were observed by MR imaging. The content of free Gd(3+), the toxic residual form, was less than 0.01%. Chelation stability of Gd-DTPA-Pullulan was estimated, and only 0.2% and 0.00045% of Gd(3+) were released from Gd-DTPA-Pullulan after 2h incubation with Ca(2+) and Fe(2+), respectively. Gd-DTPA-Pullulan displayed the extended plasma half-life (t1/2,α=0.43 h, t1/2,ß=2.32 h), much longer than 0.11h and 0.79 h of Gd-EOB-DTPA. Abdominal MR imaging showed Gd-DTPA-Pullulan maintained initial MR contrast for 30 min. The extended plasma half-life of Gd-DTPA-Pullulan probably allows the prolonged MR acquisition time in clinic with enhanced MR contrast.
Assuntos
Meios de Contraste , Gadolínio DTPA , Imageamento por Ressonância Magnética/métodos , Animais , Meios de Contraste/química , Meios de Contraste/farmacocinética , Meios de Contraste/farmacologia , Gadolínio DTPA/química , Gadolínio DTPA/farmacocinética , Gadolínio DTPA/farmacologia , Glucanos/química , Glucanos/farmacocinética , Glucanos/farmacologia , RatosRESUMO
Infantile and classical late infantile neuronal ceroid lipofuscinoses (NCL) are two recent additions to the expanding spectrum of lysosomal storage disorders caused by deficiencies in lysosomal hydrolases. They are latecomers to the lysosomal storage disorders, probably because of the heterogeneous nature of the storage material, which precluded meaningful biochemical analysis. Infantile NCL is caused by deficiency in palmitoyl-protein thioesterase, an enzyme that hydrolyzes fatty acids from cysteine residues in lipid-modified proteins. Classical late-infantile NCL is caused by a deficiency in tripeptidyl amino peptidase-I, a lysosomal peptidase that removes three amino acids from the free amino terminus of peptides or small proteins. Late-onset forms of these disorders have been described. The clinical, biochemical, and molecular genetic aspects of these two latest lysosomal storage disorders are discussed in this review. In addition, approaches to treatment and future directions for research are examined.
Assuntos
Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , Peptídeo Hidrolases/genética , Adolescente , Adulto , Idade de Início , Aminopeptidases , Criança , Pré-Escolar , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases , Humanos , Lactente , Leucócitos/ultraestrutura , Microscopia Eletrônica , Mutação , Lipofuscinoses Ceroides Neuronais/enzimologia , Lipofuscinoses Ceroides Neuronais/terapia , Polimorfismo Genético , Serina Proteases , Tioléster Hidrolases , Tripeptidil-Peptidase 1RESUMO
Exposing rare but highly malignant tumor cells that migrate from the primary tumor mass into adjacent tissue(s) or circulate in the bloodstream is critical for early detection and effective intervention(s). Here, we report on an aptamer-based strategy directed against epidermal growth factor receptor (EGFR), the most common oncogene in glioblastoma (GBM), to detect these deadly tumor cells. GBMs are characterized by diffuse infiltration into normal brain regions, and the inability to detect GBM cells renders the disease surgically incurable with a median survival of just 14.2 months. To test the sensitivity and specificity of our platform, anti-EGFR RNA aptamers were immobilized on chemically modified glass surfaces. Cells tested included primary human GBM cells expressing high levels of the wild-type EGFR, as well as genetically engineered murine glioma cells overexpressing the most common EGFR mutant (EGFRvIII lacking exons 2-7) in Ink4a/Arf-deficient astrocytes. We found that surfaces functionalized with anti-EGFR aptamers could capture both the human and murine GBM cells with high sensitivity and specificity. Our findings show how novel aptamer substrates could be used to determine whether surgical resection margins are free of tumor cells, or more widely for detecting tumor cells circulating in peripheral blood to improve early detection and/or monitoring residual disease after treatment.
Assuntos
Aptâmeros de Nucleotídeos/genética , Receptores ErbB/genética , RNA Antissenso/genética , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Astrócitos/metabolismo , Sequência de Bases , Ligação Competitiva , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Separação Celular/métodos , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , RNA Antissenso/química , RNA Antissenso/metabolismo , Ratos , Células-Tronco/metabolismo , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
PURPOSE: Telomerase activity is one of the hallmarks of cancer and is a highly relevant therapeutic target. The effects of a novel human telomerase antagonist, imetelstat, on primary human glioblastoma (GBM) tumor-initiating cells were investigated in vitro and in vivo. EXPERIMENTAL DESIGN: Tumor-initiating cells were isolated from primary GBM tumors and expanded as neurospheres in vitro. The GBM tumor-initiating cells were treated with imetelstat and examined for the effects on telomerase activity levels, telomere length, proliferation, clonogenicity, and differentiation. Subsequently, mouse orthotopic and subcutaneous xenografts were used to assess the in vivo efficacy of imetelstat. RESULTS: Imetelstat treatment produced a dose-dependent inhibition of telomerase (IC(50) 0.45 micromol/L). Long-term imetelstat treatment led to progressive telomere shortening, reduced rates of proliferation, and eventually cell death in GBM tumor-initiating cells. Imetelstat in combination with radiation and temozolomide had a dramatic effect on cell survival and activated the DNA damage response pathway. Imetelstat is able to cross the blood-brain barrier in orthotopic GBM xenograft tumors. Fluorescently labeled GBM tumor cells isolated from orthotopic tumors, following systemic administration of imetelstat (30 mg/kg every day for three days), showed approximately 70% inhibition of telomerase activity. Chronic systemic treatment produced a marked decrease in the rate of xenograft subcutaneous tumor growth. CONCLUSION: This preclinical study supports the feasibility of testing imetelstat in the treatment of GBM patients, alone or in combination with standard therapies.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Glioblastoma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Telomerase/antagonistas & inibidores , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos da radiação , Oligonucleotídeos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The neuronal ceroid lipofuscinoses (NCLs, also known collectively as Batten disease) are a group of lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in the brain and other tissues. A number of genes underlying various forms of NCL have been cloned, but the basis for the neurodegeneration in any of these is unknown. High levels of dolichol pyrophosphoryl oligosaccharides have previously been demonstrated in brain tissue from several NCL patients, but the specificity of the effect for the NCLs has been unclear. In the present study, we examine eight mouse models of lysosomal storage disorders by modern FACE and found striking lipid-linked oligosaccharide (LLO) accumulation in NCL mouse models (especially CLN1, CLN6, and CLN8 knockout or mutant mice) but not in several other lysosomal storage disorders affecting the brain. Using a mouse model of the most severe form of NCL (the PPT1 knockout mouse), we show that accumulated LLOs are not the result of a defect in LLO synthesis, extension, or transfer but rather are catabolic intermediates derived from LLO degradation. LLOs are enriched about 60-fold in the autofluorescent storage material purified from PPT1 knockoutmouse brain but comprise only 0.3% of the autofluorescent storage material by mass. The accumulation of LLOs is postulated to result from inhibition of late stages of lysosomal degradation of autophagosomes, which may be enriched in these metabolic precursors.
Assuntos
Modelos Animais de Doenças , Lipopolissacarídeos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Animais , Encéfalo/metabolismo , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos/metabolismoRESUMO
Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human PPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins. PPT1 has orthologs in many species, including lower organisms and plants, but not in Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the S. pombe Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the present study, we characterized this interesting gene (designated here as pdf1, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the pdf1 sequence. Strains bearing a deletion of the entire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid. Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (vps) mutants and vacuolar membrane H(+)-ATPase (vma) mutants in S. cerevisiae. Importantly, the Ppt1p-deficient phenotype is complemented by the human PPT1 gene. These results indicate that the function of PPT1 has been widely conserved throughout evolution and that S. pombe may serve as a genetically tractable model for the study of human infantile Batten disease.
Assuntos
Modelos Genéticos , Lipofuscinoses Ceroides Neuronais/genética , Ácido Orótico/análogos & derivados , Schizosaccharomyces/enzimologia , Tioléster Hidrolases/genética , Sequência de Aminoácidos , Clonagem Molecular , Deleção de Genes , Teste de Complementação Genética , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Ácido Orótico/farmacologia , Mutação Puntual , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Schizosaccharomyces/genética , Alinhamento de Sequência , Tioléster Hidrolases/metabolismo , Vanadatos/toxicidadeRESUMO
The CWH8 gene in Saccharomyces cerevisiae has been shown recently (Fernandez, F., Rush, J. S., Toke, D. A., Han, G., Quinn, J. E., Carman, G. M., Choi, J.-Y., Voelker, D. R., Aebi, M., and Waechter, C. J. (2001) J. Biol. Chem. 276, 41455-41464) to encode a dolichyl pyrophosphate (Dol-P-P) phosphatase associated with crude microsomal fractions. Mutations in CWH8 result in the accumulation of Dol-P-P, deficiency in lipid intermediate synthesis, defective protein N-glycosylation, and a reduced growth rate. A cDNA (DOLPP1, GenBank accession number AB030189) from mouse brain encoding a homologue of the yeast CWH8 gene is now shown to complement the defects in growth and protein N-glycosylation, and to correct the accumulation of Dol-P-P in the cwh8Delta yeast mutant. Northern blot analyses demonstrate a wide distribution of the DOLPP1 mRNA in mouse tissues. Overexpression of Dolpp1p in yeast, COS, and Sf9 cells produces substantial increases in Dol-P-P phosphatase activity but not in dolichyl monophosphate or phosphatidic acid phosphatase activities in microsomal fractions. Subcellular fractionation and immunofluorescence studies localize the enzyme encoded by DOLPP1 to the endoplasmic reticulum of COS cells. The results of protease sensitivity studies with microsomal vesicles from the lpp1Delta/dpp1Delta yeast mutant expressing DOLPP1 are consistent with Dolpp1p having a luminally oriented active site. The sequence of the DOLPP1 cDNA predicts a polypeptide with 238 amino acids, and a new polypeptide corresponding to 27 kDa is observed when DOLPP1 is expressed in yeast, COS, and Sf9 cells. This study is the first identification and characterization of a cDNA clone encoding an essential component of a mammalian lipid pyrophosphate phosphatase that is highly specific for Dol-P-P. The specificity, subcellular location, and topological orientation of the active site described in the current study strongly support a role for Dolpp1p in the recycling of Dol-P-P discharged during protein N-glycosylation reactions on the luminal leaflet of the endoplasmic reticulum in mammalian cells.