RESUMO
The clinical manifestations of sporadic amyotrophic lateral sclerosis (ALS) vary widely. However, the current classification of ALS is mainly based on clinical presentations, while the roles of electrophysiological and biomedical biomarkers remain limited. Herein, we investigated a group of patients with sporadic ALS and an ALS mouse model with superoxide dismutase 1 (SOD1)/G93A transgenes using nerve excitability tests (NET) to investigate axonal membrane properties and chemical precipitation, followed by enzyme-linked immunosorbent assay analysis to measure plasma misfolded protein levels. Six of 19 patients (31.6%) with sporadic ALS had elevated plasma misfolded SOD1 protein levels. In sporadic ALS patients, only those with elevated misfolded SOD1 protein levels showed an increased inward rectification in the current-threshold (I/V) curve and an increased threshold reduction in the hyperpolarizing threshold electrotonus (TE) in the NET study. Two familial ALS patients with SOD1 mutations also exhibited similar electrophysiological patterns of NET. For patients with sporadic ALS showing significantly increased inward rectification in the I/V curve, we noted an elevation in plasma misfolded SOD1 level, but not in total SOD1, misfolded C9orf72, or misfolded phosphorylated TDP43 levels. Computer simulations demonstrated that the aforementioned axonal excitability changes are likely associated with an increase in hyperpolarization-activated cyclic nucleotide-gated (HCN) current. In SOD1/G93A mice, NET also showed an increased inward rectification in the I/V curve, which could be reversed by a single injection of the HCN channel blocker, ZD7288. Daily treatment of SOD1/G93A mice with ZD7288 partially prevented the early motor function decline and spinal motor neuron death. In summary, sporadic ALS patients with elevated plasma misfolded SOD1 exhibited similar patterns of motor axonal excitability changes as familial ALS patients and ALS mice with mutant SOD1 genes, suggesting the existence of SOD1-associated sporadic ALS. The observed NET pattern of increased inward rectification in the I/V curve was attributable to an elevation in the HCN current in SOD1-associated ALS.
RESUMO
Programmed death-ligand 1 (PD-L1) is an inhibitory transmembrane protein that can prevent autoimmune response. Upregulated PD-L1 serves as a predictive biomarker for patients who may respond well to immune checkpoint therapies. However, variable associations of PD-L1 level with prognoses have been reported. In this study, a short peptide sequence corresponding to PD-L1 amino acids 172-187 (from the extracellular Ig-like C-type domain, and with high predicted antigenicity and hydrophilicity) was used to generate a monoclonal antibody (mAb). The resultant PD-L1 mAb, clone HC16, was examined for binding specificity and reactivity in cancer cell-lines, as assessed by immunocytochemical, immunoblotting, and co-immunoprecipitation. The potential diagnostic and clinical applicability of clone HC16 was further tested using malignant tissue arrays derived from various cancer types analyzed with an automated immunohistochemical (IHC) staining platform. Additionally, tumor samples from patients diagnosed with non-small cell lung cancer (NSCLC) were analyzed by western blotting. Clone HC16 showed obvious staining activity in lung and breast cancer tissues. Interestingly, we observed that PD-L1 level was negatively associated with clinical stage in NSCLC. Strong PD-L1 expression tended to be found in patients diagnosed with bronchioloalveolar carcinoma (BAC). These results demonstrate that clone HC16 harbors good target specificity and is suitable for further development in diagnostic tools to assess PD-L1 expression in human tissues. In addition, our findings also suggest a role for PD-L1 in a non-invasive subtype of lung cancer.
Assuntos
Anticorpos Monoclonais/química , Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Regulação para Cima , Células A549 , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cromossomos Artificiais Bacterianos , Epitopos/química , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Reprodutibilidade dos TestesRESUMO
Tilapia piscidin (TP) 4 is an antimicrobial peptide derived from Nile tilapia (Oreochromis niloticus), which shows broad-spectrum antibacterial activity and excellent cancer-killing ability in vitro and in vivo. Like many other antimicrobial peptides, TP4 treatment causes mitochondrial toxicity in cancer cells. However, the molecular mechanisms underlying TP4 targeting of mitochondria remain unclear. In this study, we used a pull-down assay on A549 cell lysates combined with LC-MS/MS to discover that TP4 targets adenine nucleotide translocator (ANT) 2, a protein essential for adenine nucleotide exchange across the inner membrane. We further showed that TP4 accumulates in mitochondria and colocalizes with ANT2. Moreover, molecular docking studies showed that the interaction requires Phe1, Ile2, His3, His4, Ser11, Lys14, His17, Arg21, Arg24 and Arg25 residues in TP4 and key residues within the cavity of ANT2. These findings suggest a mechanism by which TP4 may induce mitochondrial dysfunction to disrupt cellular energy metabolism.
Assuntos
Translocador 2 do Nucleotídeo Adenina/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Ciclídeos/metabolismo , Proteínas de Peixes/farmacologia , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Células A549 , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/metabolismo , Humanos , Células MCF-7 , Microscopia Confocal , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Ligação ProteicaRESUMO
Hepcidin regulates iron homeostasis and host-defense mechanisms, while the hepcidin-like protein, Tilapia hepcidin (TH)2-3, functions as an antimicrobial peptide (AMP). Since AMP dietary supplements may be used as alternatives to antibiotics in livestock, we tested the effects of recombinant (r)TH2-3 as a dietary supplement in grouper aquaculture. rTH2-3 was produced by a Pichia pastoris expression system and exhibited thermostability and broad-spectrum antimicrobial activity. The feed conversion ratio and feed efficiency were determined in Epinephelus lanceolatus (grouper) fed with rTH2-3-supplemented diet for 28 days. In addition, grouper showed enhanced superoxide dismutase (SOD) activity after rTH2-3 feeding compared to regular-diet-fed fish. Gut microbiota analysis revealed that microbial diversity was enhanced by feeding grouper with 1% rTH2-3. After challenging grouper with Vibrio alginolyticus, differential regulation of immune-related genes in the liver and spleen was observed between the TH2-3 and regular-diet groups, including for genes associated with antimicrobial and pro-inflammatory functions, complement components, and major histocompatibility complex (Mhc). These findings suggest that overall immunity was improved. Thus, our results suggest long-term supplementation with rTH2-3 may be beneficial for aquacultured grouper. The beneficial effects of the supplement are likely based on changes in the commensal microbial community as well as immunomodulation.
Assuntos
Bass/imunologia , Bass/microbiologia , Suplementos Nutricionais , Microbioma Gastrointestinal/efeitos dos fármacos , Hepcidinas/farmacologia , Imunomodulação/efeitos dos fármacos , Tilápia/metabolismo , Ração Animal , Animais , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bass/genética , Bass/crescimento & desenvolvimento , Comportamento Alimentar/efeitos dos fármacos , Fermentação , Regulação da Expressão Gênica/efeitos dos fármacos , Metagenômica , Testes de Sensibilidade Microbiana , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Baço/metabolismo , TemperaturaRESUMO
Non-small cell lung cancer (NSCLC) is among the leading causes of human mortality. One reason for high rates of NSCLC mortality is that drug resistance is a major problem for both conventional chemotherapies and less-toxic targeted therapies. Thus, novel mechanistic insights into disease pathogenesis may benefit the development of urgently needed therapies. Here we show that FBJ murine osteosarcoma viral oncogene homolog B (FOSB) was induced by an antimicrobial peptide, tilapia piscidin-4 (TP4), through the dysregulation of mitochondrial Ca2+ homeostasis in NSCLC cells. Transcriptomic, chromatin immunoprecipitation quantitative PCR, and immunocytochemical studies reveal that protocadherin-ß13 (PCDHB13) as a target of FOSB that was functionally associated with microtubule. Overexpression of either PCDHB13 or FOSB attenuated NSCLC growth and survival in vitro and in vivo. Importantly, downregulation of both FOSB and PCDHB13 was observed in NSCLC patients and was negatively correlated with pathological grade. These findings introduce the FOSBâ»PCDHB13 axis as a novel tumor suppressive pathway in NSCLC.
RESUMO
Non-small cell lung cancer (NSCLC) is among the leading causes of human mortality due to a lack of effective treatments. Conventional chemotherapies affect healthy cells and cause multidrug resistance, while tumors may eventually develop resistance to less-toxic targeted therapies. Thus, the need to develop novel therapies for NSCLC is urgent. Here, we show that Nile tilapia-derived Tilapia piscidin (TP) 4 is cytotoxic to a panel of NSCLC cells with different genetic profiles. We observed that TP4 triggers NSCLC cell death through the necrosis and combining TP4 with potent Epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKI)s, Erlotinib, and Gefitinib, improved drug responses in EGFR-mutated NSCLC cells, but not in EGFR-wild-type NSCLC cells. This work provides novel insights into potential NSCLC treatments, which may utilize antimicrobial peptide TP4 as monotherapy or in combination with EGFR-TKIs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ciclídeos , Proteínas de Peixes/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/uso terapêutico , Gefitinibe/farmacologia , Humanos , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Some antimicrobial peptides (AMPs) exhibit anti-cancer activity, acting on cancer cells either by causing membrane lysis or via intracellular effects. While intracellular penetration of AMPs has been shown to cause cancer cell death, the mechanisms of toxicity remain largely unknown. Here we show that a tilapia-derived AMP, Tilapia piscidin (TP) 4, penetrates intracellularly and targets the microtubule network. A pull-down assay identified α-Tubulin as a major interaction partner for TP4, and molecular docking analysis suggested that Phe1, Ile16, and Arg23 on TP4 are required for the interaction. TP4 treatment in A549 cells was found to disrupt the microtubule network in cells, and mutation of the essential TP4 residues prevented microtubule depolymerization in vitro. Importantly, the TP4 mutants also showed decreased cytotoxicity in A549 cells, suggesting that microtubule disruption is a major mechanistic component of TP4-mediated death in lung carcinoma cells.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Ciclídeos , Proteínas de Peixes/farmacologia , Microtúbulos/efeitos dos fármacos , Células A549 , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Peixes/isolamento & purificação , Humanos , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/isolamento & purificação , Moduladores de Tubulina/farmacologiaRESUMO
Artemia are often used as a live feed for fry in aquaculture. We have previously demonstrated that supplementing adult zebrafish feed with Artemia, which express an Epinephelus coioides-derived antimicrobial peptide, Epinecidin-1 (Epi-1), protects against bacterial infection. Thus, Artemia may serve as a bioreactor for producing biofunctional molecules. However, the application of Epi-1 transgenic Artemia in larval aquaculture of commercial fish species has not been investigated. Here we used a Tol2-transposon system to generate stable Epi-1 expressing Artemia. Nile tilapia (Oreochromis niloticus) fry were then fed with decapsulated transgenic cysts and acutely challenged with Gram-positive Streptococcus iniae or Gram-negative Vibrio vulnificus (204). Survival analysis revealed that tilapia fry fed with Epi-1 transgenic cysts were resistant to acute bacterial infection. Immune-related gene expression profiling showed that S. iniae and V. vulnificus inoculations produced distinct immunomodulatory effects in the tilapia fry. Upon S. iniae infection, tilapia fry fed on control diet exhibited an immune response dominated by Tlr-7/MyD88, wherein Tnf-α, Il-8 and Cxcl-10 expression were all induced; conversely, the tilapia fry fed with Epi-1 transgenic cysts showed a Tlr-2/Tlr-5-dominant immune response, marked by the induction of Il-1ß, Il-8 and Il-12 expression. However, after V. vulnificus (204) infection control fry exhibited a Tlr-2/MyD88/Traf-6-dominant response with activation of Tnf-α and Il-8 expression; meanwhile tilapia fry fed on Epi-1 transgenic cyst showed a dominant Tlr-2/Tlr-5-mediated immune response, including induction of Il-1ß, Il-8, Il-12, and Cxcl-10 expression. These findings suggest that feeding larval fish fry with Epi-1 transgenic Artemia cysts confers enhanced immunity toward bacterial challenge. Epi-1 transgenic cysts should therefore be considered as a potential functional feed for larval aquaculture.
Assuntos
Ração Animal , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Artemia , Infecções Bacterianas/veterinária , Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Doença Aguda , Animais , Animais Geneticamente Modificados , Aquicultura , Infecções Bacterianas/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata , Larva , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus iniae , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio vulnificusRESUMO
Streptococcus agalactiae infection is one of the most significant bacterial diseases in tilapia aquaculture. Identification of immune-related genes associated with Streptococcus agalactiae infection may provide a basis for breeding selection or therapeutics to augment disease resistance. Therefore, we utilized transcriptome profiling to study the host response in tilapia following Streptococcus agalactiae infection. Based on GO and KEGG enrichment analyses, we found that differentially expressed genes are widely involved in immune-related pathways, including the induction of antimicrobial peptides. Moreover, the main components of two immune-related pathways (Toll-like receptor signaling and leukocyte transendothelial migration) and four environmental information processing pathways (TNF, PI3K-Akt, Jak-STAT and MAPK) were identified. Finally, a time-course expression profile for several of the identified transcripts including tilapia piscidin 3 (TP3), tilapia piscidin 4 (TP4), TLR2, TLR5, MyD88, TRAF6, p38, and interleukin components was performed by qRT-PCR. Collectively, these results provide a starting point to study molecular mechanisms of tilapia immune response to Streptococcus agalactiae infection and may be applied as a basis for developing disease resistant strains by breeding selection.
Assuntos
Ciclídeos , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Perfilação da Expressão Gênica/veterinária , Transdução de Sinais , Infecções Estreptocócicas/veterinária , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/fisiologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Triple-negative breast cancer (TNBC) currently lacks a suitable therapeutic candidate and is thus difficult to treat. Here, we report that a cationic antimicrobial peptide (CAP), tilapia piscidin 4 (TP4), which was derived from Nile tilapia (Oreochromis niloticus), is selectively toxic to TNBC. TP4 acts by inducing an AP-1 protein called FOSB, the expression of which is negatively associated with the pathological grade of TNBC. We show that TP4 is bound to the mitochondria where it disrupts calcium homeostasis and activates FOSB. FOSB overexpression results in TNBC cell death, whereas inhibition of calcium signaling eliminates FOSB induction and blocks TP4-induced TNBC cell death. Both TP4 and anthracyclines strongly induced FOSB, particularly in TNBC, indicating that FOSB may be suitable as a biomarker of drug responses. This study thus provides a novel therapeutic approach toward TNBC through FOSB induction.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Biomarcadores Tumorais/metabolismo , Cálcio/metabolismo , Morte Celular , Linhagem Celular Tumoral , Ciclídeos , Feminino , Proteínas de Peixes/farmacologia , Perfilação da Expressão Gênica , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Necrose , Transplante de Neoplasias , Domínios Proteicos , Fator de Transcrição AP-1/metabolismoRESUMO
Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by a deficiency of survival motor neuron (SMN) protein. In this study, we evaluated the efficacy of intermittent transient hypothermia in a mouse model of SMA. SMA mice were exposed to ice for 50 s to achieve transient hypothermia (below 25°C) daily beginning on postnatal day 1. Neonatal SMA mice (Smn(-/-)SMN2(+/-)) who received daily transient hypothermia exhibited reduced motor neuron degeneration and muscle atrophy and preserved the architecture of neuromuscular junction when compared with untreated controls at day 8 post-treatment. Daily hypothermia also prolonged the lifespan, increased body weight and improved motor coordination in SMA mice. Quantitative polymerase chain reaction and western blot analyses showed that transient hypothermia led to an increase in SMN transcript and protein levels in the spinal cord and brain. In in vitro studies using an SMN knockdown motor neuron-like cell-line, transient hypothermia increased intracellular SMN protein expression and length of neurites, confirming the direct effect of hypothermia on motor neurons. These data indicate that the efficacy of intermittent transient hypothermia in improving outcome in an SMA mouse model may be mediated, in part, via an upregulation of SMN levels in the motor neurons.
Assuntos
Hipotermia Induzida/métodos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 µg/100 µl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.
Assuntos
Animais Geneticamente Modificados/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/imunologia , Vibrioses/veterinária , Peixe-Zebra , Análise de Variância , Animais , Animais Geneticamente Modificados/genética , Artemia/genética , Artemia/metabolismo , Dieta/veterinária , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/veterinária , Eletroporação/veterinária , Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sobrevida , Vibrioses/imunologiaRESUMO
Spinal muscular atrophy is a progressive motor neuron disease caused by a deficiency of survival motor neuron. In this study, we evaluated the efficacy of intravenous administration of a recombinant adeno-associated virus (AAV1) vector encoding human insulin-like growth factor-1 (IGF-1) in a severe mouse model of spinal muscular atrophy. Measurable quantities of human IGF-1 transcripts and protein were detected in the liver (up to 3 months postinjection) and in the serum indicating that IGF-1 was secreted from the liver into systemic circulation. Spinal muscular atrophy mice administered AAV1-IGF-1 on postnatal day 1 exhibited a lower extent of motor neuron degeneration, cardiac and muscle atrophy as well as a greater extent of innervation at the neuromuscular junctions compared to untreated controls at day 8 posttreatment. Importantly, treatment with AAV1-IGF-1 prolonged the animals' lifespan, increased their body weights and improved their motor coordination. Quantitative polymerase chain reaction and western blot analyses showed that AAV1-mediated expression of IGF-1 led to an increase in survival motor neuron transcript and protein levels in the spinal cord, brain, muscles, and heart. These data indicate that systemically delivered AAV1-IGF-1 can correct several of the biochemical and behavioral deficits in spinal muscular atrophy mice through increasing tissue levels of survival motor neuron.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Fator de Crescimento Insulin-Like I/genética , Atrofia Muscular Espinal/fisiopatologia , Atrofia Muscular Espinal/terapia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Humanos , Injeções Intravenosas , Fator de Crescimento Insulin-Like I/administração & dosagem , Fígado/metabolismo , Camundongos , Atrofia Muscular Espinal/sangue , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Resultado do TratamentoRESUMO
Pardaxin is a cationic antimicrobial peptide derived from Red Sea Moses sole. Previous studies have shown that pardaxin selectively triggers the death of cancer cells, initiating the development of a pardaxin-based cancer vaccine; however, the underlying mechanism by which pardaxin kills cancer cells has not yet been elucidated. Here, we demonstrate that this mechanism involves endoplasmic reticulum (ER) targeting and c-FOS induction. Transcriptiome analysis of pardaxin-treated HT-1080 cells revealed induction of the gene encoding c-FOS, an AP-1 transcription factor. Pardaxin mediates cell death by activating c-FOS, but not other AP-1 transcription factors. Overexpression of c-FOS caused a dramatic increase in cell death, while knockdown of c-FOS induced pardaxin resistance; such effects were observed in both an in vitro cell model and an in vivo xenograft tumor model. Treatment with pardaxin also increased the level of calcium, and blockage of cellular calcium signaling disrupted pardaxin-induced cell death. Immunocytochemistry was used to demonstrate targeting of pardaxin to the endoplasmic reticulum, but not to the Golgi apparatus or mitochondria. Importantly, pardaxin treatment or c-FOS overexpression induced cell death in diverse cancer cell lines, indicating that pardaxin and c-FOS may possess therapeutic potential for use in cancer treatment.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Retículo Endoplasmático/metabolismo , Venenos de Peixe/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Venenos de Peixe/química , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Proximal spinal muscular atrophy (SMA), a neurodegenerative disorder that causes infant mortality, has no effective treatment. Sodium vanadate has shown potential for the treatment of SMA; however, vanadate-induced toxicity in vivo remains an obstacle for its clinical application. We evaluated the therapeutic potential of sodium vanadate combined with a vanadium detoxification agent, L-ascorbic acid, in a SMA mouse model. METHODS: Sodium vanadate (200 µM), L-ascorbic acid (400 µM), or sodium vanadate combined with L-ascorbic acid (combined treatment) were applied to motor neuron-like NSC34 cells and fibroblasts derived from a healthy donor and a type II SMA patient to evaluate the cellular viability and the efficacy of each treatment in vitro. For the in vivo studies, sodium vanadate (20 mg/kg once daily) and L-ascorbic acid (40 mg/kg once daily) alone or in combination were orally administered daily on postnatal days 1 to 30. Motor performance, pathological studies, and the effects of each treatment (vehicle, L-ascorbic acid, sodium vanadate, and combined treatment) were assessed and compared on postnatal days (PNDs) 30 and 90. The Kaplan-Meier method was used to evaluate the survival rate, with P < 0.05 indicating significance. For other studies, one-way analysis of variance (ANOVA) and Student's t test for paired variables were used to measure significant differences (P < 0.05) between values. RESULTS: Combined treatment protected cells against vanadate-induced cell death with decreasing B cell lymphoma 2-associated X protein (Bax) levels. A month of combined treatment in mice with late-onset SMA beginning on postnatal day 1 delayed disease progression, improved motor performance in adulthood, enhanced survival motor neuron (SMN) levels and motor neuron numbers, reduced muscle atrophy, and decreased Bax levels in the spinal cord. Most importantly, combined treatment preserved hepatic and renal function and substantially decreased vanadium accumulation in these organs. CONCLUSIONS: Combined treatment beginning at birth and continuing for 1 month conferred protection against neuromuscular damage in mice with milder types of SMA. Further, these mice exhibited enhanced motor performance in adulthood. Therefore, combined treatment could present a feasible treatment option for patients with late-onset SMA.
Assuntos
Ácido Ascórbico/administração & dosagem , Destreza Motora/efeitos dos fármacos , Debilidade Muscular/tratamento farmacológico , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular/tratamento farmacológico , Vanadatos/administração & dosagem , Adulto , Animais , Células Cultivadas , Progressão da Doença , Quimioterapia Combinada , Estudos de Viabilidade , Feminino , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Destreza Motora/fisiologia , Debilidade Muscular/patologia , Atrofia Muscular/patologia , Atrofia Muscular Espinal/patologiaRESUMO
Spinal muscular atrophy (SMA), a genetic neurodegenerative disorder, is caused by mutations or deletions in the survival of motor neuron 1 (SMN1) gene that result in SMN deficiency. SMN deficiency impairs microtubule networks in Smn-deficient cells and in SMA-like motor neuron cultures. Microtubule defects can be restored by knockdown of the stathmin gene (Stmn), which is upregulated in SMA. However, whether in vivo reduction of stathmin levels could improve the pathology of SMA has not been investigated. Here we generated SMA-like mice in a Stmn knockout (KO) background through a series of genetic crosses. Analyses of motor performance and histology showed that heterozygous StmnKO (Stmn(+/-)) but not homozygous StmnKO (Stmn(-/-)) ameliorates some SMA defects, with increased microtubule densities in sciatic axons, improved motor performance, enhanced NMJ maturation, and mitigated neuroinflammation. However, Stmn deletion does not prolong the lifespan of SMA-like mice, suggesting that stathmin dysregulation and microtubule disruption are not a cause but rather a consequence of SMA pathology. This work demonstrates that limiting the amount of stathmin in SMA-like mice is effective in reducing their neuromuscular defects, whereas induced aberrant expression of stathmin in SMA-like animals is detrimental.
Assuntos
Longevidade/genética , Atrofia Muscular Espinal/metabolismo , Estatmina/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Regulação para Baixo , Camundongos , Camundongos Knockout , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Estatmina/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Regulação para CimaRESUMO
Proximal spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by deficiency of the ubiquitous Survival of Motor Neuron (SMN) protein. SMN has been shown to be transported in granules along the axon and moved through cytoskeletal elements. However, the role and nature of SMN granules are still not well characterized. Here, using immunocytochemical methods and time-lapse studies we show that SMN granules colocalize with the Golgi apparatus in motor neuron-like NSC34 cells. Electron microscopy clearly revealed that SMN granules are transported into the Golgi stack and aggregate in the trans-Golgi apparatus. SMN granules are characterized as either coated or un-coated and behave like regulated secretory granules. Treatment of cells with monensin to disrupt Golgi-mediated granule secretion decreased SMN expression in neurites and caused growth cone defects similar to those seen in SMN knockdown cells. Knockdown of Cop-α, the protein that coats vesicles transporting proteins between the Golgi compartments, caused SMN granule accumulation in the Golgi apparatus. In addition to the well-studied role of SMN in small nuclear ribonucleoprotein (SnRNP) assembly, this work links SMN granules with the Golgi network and thus sheds light on Golgi-mediated SMN granule transport.
Assuntos
Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Complexo de Golgi/metabolismo , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/fisiologia , Animais , Western Blotting , Núcleo Celular/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Imunofluorescência , Técnicas Imunoenzimáticas , Camundongos , Microscopia Eletrônica de Transmissão , Neurônios Motores/citologia , Neuritos/metabolismo , Transporte Proteico , RNA Interferente Pequeno/genética , Proteína 1 de Sobrevivência do Neurônio Motor/antagonistas & inibidores , Imagem com Lapso de TempoRESUMO
The efficacy of administering a recombinant adeno-associated virus (AAV) vector encoding human IGF-1 (AAV2/1-hIGF-1) into the deep cerebellar nucleus (DCN) of a type III SMA mouse model was evaluated. High levels of IGF-1 transcripts and protein were detected in the spinal cord at 2 months post-injection demonstrating that axonal connections between the cerebellum and spinal cord were able to act as conduits for the viral vector and protein to the spinal cord. Mice treated with AAV2/1-hIGF-1 and analyzed 8 months later showed changes in endogenous Bax and Bcl-xl levels in spinal cord motor neurons that were consistent with IGF-1-mediated anti-apoptotic effects on motor neurons. However, although AAV2/1-hIGF-1 treatment reduced the extent of motor neuron cell death, the majority of rescued motor neurons were non-functional, as they lacked axons that innervated the muscles. Furthermore, treated SMA mice exhibited abnormal muscle fibers, aberrant neuromuscular junction structure, and impaired performance on motor function tests. These data indicate that although CNS-directed expression of IGF-1 could reduce motor neuron cell death, this did not translate to improvements in motor function in an adult mouse model of type III SMA.
Assuntos
Morte Celular/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/uso terapêutico , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/terapia , Animais , Morte Celular/fisiologia , Cerebelo/efeitos dos fármacos , Cerebelo/patologia , Cerebelo/fisiopatologia , Terapia Genética , Vetores Genéticos , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Atividade Motora/fisiologia , Neurônios Motores/patologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
Spinal muscular atrophy (SMA), a motor neuron degeneration disorder, is caused by either mutations or deletions of survival motor neuron 1 (SMN1) gene which result in insufficient SMN protein. Here, we describe a potential link between stathmin and microtubule defects in SMA. Stathmin was identified by screening Smn-knockdown NSC34 cells through proteomics analysis. We found that stathmin was aberrantly upregulated in vitro and in vivo, leading to a decreased level of polymerized tubulin, which was correlated with disease severity. Reduced microtubule densities and beta(III)-tubulin levels in distal axons of affected SMA-like mice and an impaired microtubule network in Smn-deficient cells were observed, suggesting an involvement of stathmin in those microtubule defects. Furthermore, knockdown of stathmin restored the microtubule network defects of Smn-deficient cells, promoted axon outgrowth and reduced the defect in mitochondria transport in SMA-like motor neurons. We conclude that aberrant stathmin levels may play a detrimental role in SMA; this finding suggests a novel approach to treating SMA by enhancing microtubule stability.
Assuntos
Microtúbulos/metabolismo , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/metabolismo , Estatmina/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Western Blotting , Linhagem Celular , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Microtúbulos/patologia , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Oligonucleotídeos/genética , Plasmídeos/genéticaRESUMO
Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in Apc(Min/+) mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4-ephrin-A and EphB4-ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3-ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes' stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression re-organized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical actin cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased transwell migration, increased apoptosis and Ca(2+)-dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear beta-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL-Rac1, a known epithelial-to-mesenchymal transition signaling pathway. Additionally, cross talk from Wnt signaling potentiated the restoration of epithelial cell polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3-ephrin-B interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect contributes to EphB3-mediated tumor suppression.