RESUMO
Background and Purpose: CAPN1 (calpain1)an intracellular Ca2+-regulated cysteine proteasecan be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined. Methods: In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucosedeprived primary neuron in vitro model. Results: CAPN1 was activated in the cortex of permanent middle cerebral occlusionoperated rats and oxygen-glucose deprivationexposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage. Conclusions: The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemiamediated autophagy-lysosomal pathway defects and neuronal damage.
Assuntos
Autofagia/fisiologia , Isquemia Encefálica/metabolismo , Calpaína/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Neurônios/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Masculino , Neurônios/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
We previously reported that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, significantly ameliorated Alzheimer's disease (AD)-associated cognitive defects in APP/PS1 and SAMP8 mice by inhibiting Aß aggregation and tau hyperphosphorylation, suggesting a potential therapeutic effect of PF11 in the treatment of AD. In the present study we further evaluated the therapeutic effects of PF11 on relieving cognitive impairment in a rat model of sporadic AD (SAD). SAD was induced in rats by bilateral icv infusion of streptozotocin (STZ, 3 mg/kg). The rats were treated with PF11 (2, 4, 8 mg·kg-1·d-1, ig) or a positive control drug donepezil (5 mg·kg-1·d-1, ig) for 4 weeks. Their cognitive function was assessed in the nest building, Y-maze, and Morris water maze tests. We showed that STZ icv infusion significantly affected the cognitive function, tau phosphorylation, and insulin signaling pathway in the hippocampus. Furthermore, STZ icv infusion resulted in significant upregulation of the calpain I/cyclin-dependent protein kinase 5 (CDK5) signaling pathway in the hippocampus. Oral administration of PF11 dose-dependently ameliorated STZ-induced learning and memory defects. In addition, PF11 treatment markedly reduced the neuronal loss, protected the synapse structure, and modulated STZ-induced expression of tau phosphorylation by regulating the insulin signaling pathway and calpain I/CDK5 signaling pathway in the hippocampus. Donepezil treatment exerted similar beneficial effects in STZ-infused rats as the high dose of PF11 did. This study highlights the excellent therapeutic potential of PF11 in managing AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Disfunção Cognitiva/tratamento farmacológico , Ginsenosídeos/farmacologia , Proteínas tau/metabolismo , Doença de Alzheimer/induzido quimicamente , Animais , Calpaína/metabolismo , Pareamento Cromossômico , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/ultraestrutura , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Teste do Labirinto Aquático de Morris/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , EstreptozocinaRESUMO
Pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, has been reported to have anti-inflammatory properties, but the effects of PF11 on acute lung inflammation were unknown. The present study aimed to investigate the protective effects and potential mechanisms of PF11 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in male BALB/c mice. After being treated with PF11 (3, 10, and 30 mg/kg, intravenous) once a day for 3 consecutive days, the mice were challenged by intratracheal instillation of LPS, and then their lung tissues and bronchoalveolar lavage fluid (BALF) were collected for further analysis. The results showed that PF11 attenuated LPS-induced ALI, with alleviated histopathological damage, decreased lung wet/dry weight ratio, and reduced protein concentration and inflammatory cells number in BALF. Moreover, PF11 reversed the LPS-induced increases of mRNA expression and protein levels of interleukin-6, tumor necrosis factor-α, and interleukin-1ß. Meanwhile, PF11 decreased LPS-induced myeloperoxidase activity and neutrophil infiltration in lung tissue by reducing the expression of macrophage inflammatory protein-2 and intercellular adhesion molecule-1, as well as enhanced neutrophil clearance by accelerating neutrophils apoptosis and their phagocytosis by alveolar macrophages. In conclusion, these results indicated that PF11 significantly attenuated LPS-induced ALI through suppressing neutrophil infiltration and accelerating neutrophil clearance, suggesting its potential in the treatment of ALI.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Movimento Celular/efeitos dos fármacos , Ginsenosídeos/farmacologia , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Ginsenosídeos/uso terapêutico , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Mounting attention has been focused on defects in macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemia. TFEB (transcription factor EB)-mediated induction of ALP has been recently considered as the common mechanism in ameliorating the pathological lesion of myocardial ischemia and neurodegenerative diseases. Here we explored the vital role of TFEB in permanent middle cerebral artery occlusion (pMCAO)-mediated dysfunction of ALP and ischemic insult in rats. The results showed that ALP function was first enhanced in the early stage of the ischemic process, especially in neurons of the cortex, and this was accompanied by increased TFEB expression and translocation to the nucleus, which was mediated at least in part through activation by PPP3/calcineurin. At the later stages of ischemia, a gradual decrease in the level of nuclear TFEB was coupled with a progressive decline in lysosomal activity, accumulation of autophagosomes and autophagy substrates, and exacerbation of the ischemic injury. Notably, neuron-specific overexpression of TFEB significantly enhanced ALP function and rescued the ischemic damage, starting as early as 6 h and even lasting to 48 h after ischemia. Furthermore, neuron-specific knockdown of TFEB markedly reversed the activation of ALP and further aggravated the neurological deficits and ischemic outcome at the early stage of pMCAO. These results highlight neuronal-targeted TFEB as one of the key players in the pMCAO-mediated dysfunction of ALP and ischemic injury, and identify TFEB as a promising target for therapies aimed at neuroprotection in cerebral ischemia. Abbreviations: AAV, adeno-associated virus; AIF1/IBA1, allograft inflammatory factor 1; ALP, autophagy-lysosomal pathway; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; CsA, cyclosporin A; GFAP, glial fibrillary acidic protein; LAMP, lysosomal-associated membrane protein; LC3, microtubule-associated protein 1 light chain 3; MAP2, microtubule-associated protein 2; mNSS, modified Neurological Severity Score; MTOR, mechanistic target of rapamycin kinase; OGD, oxygen and glucose deprivation; pMCAO, permanent middle cerebral artery occlusion; RBFOX3/NeuN, RNA binding fox-1 homolog 3; SQSTM1, sequestosome1; TFEB, transcription factor EB; TTC, 2,3,5-triphenyltetrazolium chloride.
Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Isquemia Encefálica/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Lisossomos/metabolismo , Neurônios/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Encéfalo/enzimologia , Encéfalo/patologia , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/enzimologia , Isquemia Encefálica/patologia , Calcineurina/metabolismo , Núcleo Celular/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/cirurgia , Lisossomos/enzimologia , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
There is growing evidence that uridine may act as an endogenous neuromodulator with a potential signaling role in the central nervous system in addition to its function in pyrimidine metabolism. We previously found that acute morphine treatment significantly increased uridine release in the dorsal striatum of mice, indicating that uridine may contribute to morphine-induced neurobehavioral changes. In the present study, we analyzed the mechanism involved in morphine-induced uridine release and the role of uridine in morphine-induced neurobehavioral changes. Uridine release in the dorsal striatum of mice was assessed by in vivo microdialysis coupled with high performance liquid chromatography (HPLC) after morphine treatment. Western blotting and immunofluorescence were used to evaluate the expression of uridine-related proteins. Morphine-induced neurobehavioral changes were assessed by locomotor activity, behavioral sensitization and conditioned place preference (CPP) test. The expression of NT5E, an extracellular enzyme involved in formation of nucleosides, including uridine, was specifically knocked down in the dorsal striatum of mice using adeno-associated virus (AAV)-mediated short hairpin RNA (shRNA). The results indicated that both acute and chronic morphine administration significantly increased uridine release in the dorsal striatum, and this was associated with upregulation of NT5E but not other uridine-related proteins. Inhibition of NT5E with APCP or shRNA markedly inhibited morphine-induced uridine release in the dorsal striatum and related neurobehavioral changes, including hyperlocomotor activity, behavioral sensitization and CPP. Our data give a better understanding of the contribution of NT5E to morphine-induced uridine release and neurobehavioral changes, and identify NT5E as a potential target for treating morphine abuse.
Assuntos
5'-Nucleotidase/metabolismo , Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Corpo Estriado/metabolismo , Locomoção/efeitos dos fármacos , Morfina/farmacologia , Uridina/metabolismo , 5'-Nucleotidase/antagonistas & inibidores , Animais , Fosfatos de Dinucleosídeos/farmacologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Masculino , Camundongos , Microdiálise , RNA Interferente Pequeno/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Autophagy, a cellular survival mechanism, is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy, thus decreasing drug-induced apoptosis. Disulfiram (DSF), a drug widely used to control alcoholism, possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models, and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells. The present study is designed to investigate whether DSF/Cu can induce autophagy in NSCLC cells and to determine the relationship between autophagy and apoptosis. First, we observed that DSF/Cu induced significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells, accompanied by the formation of vacuoles in the cytoplasm. Next, we investigated the levels of autophagic markers, including LC3II (microtubule-associated protein 1 light chain 3), ATG5 and p62 with or without chloroquine by Western blot. In addition, we observed co-localization of LC3 and the lysosomal protein LAMP2 (lysosome-associated membrane protein 2) after treatment with DSF/Cu. The results showed that DSF/Cu induced autophagy. Finally, we demonstrated that DSF/Cu-induced apoptosis was greatly enhanced when autophagy was suppressed with Atg5 siRNA or 3-MA in NSCLC cells. This synergistic effect of DSF/Cu and 3-MA was further confirmed in the NSCLC xenograft model. Taken together, our results show that DSF/Cu stimulates autophagy in NSCLC cells, which is an impediment to DSF/Cu-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Cobre/química , Dissulfiram/química , Dissulfiram/farmacologia , Neoplasias Pulmonares/patologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary renal lymphoma (PRL) is a rare disease, with no more than 70 cases reported in the literature. The present study reports the case of a 70-year-old woman with PRL. The patient was asymptomatic, however, a mass on the right kidney was identified incidentally during routine physical examination. Computed tomography revealed a mass in the right kidney that was 3.6 cm in diameter. Subsequently, right nephrectomy was performed. The histological evaluation of the nephrectomy specimen showed diffuse large B-cell non-Hodgkin's lymphoma. The patient was treated with 6-8 cycles of a cyclophosphamide, epirubicin, vindesine and dexamethasone regimen. Follow-up examination performed after 2 months of treatment revealed no evidence of local recurrence. The present study also reviewed 49 cases of PRL that have been reported since 1989. It was found that a shorter survival time was experienced by patients with bilateral PRL (mean, 21 months) compared with unilateral PRL (mean, 68 months). A shorter survival time was also experienced by patients who were treated with chemotherapy only (mean, 15.8 months) compared with those who were treated with combination chemotherapy and surgery (mean, 49.4 months).
RESUMO
In order to improve the delivery efficiency of microRNA (miRNA or miR)-145, the present study examined several factors which may affect cationic liposome (CL)-based transfection, including the hydration medium used for the preparation of liposomes, the quantity of the plasmid, the molar ratio of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol (chol), or DOTAP/chol, and the weight ratio of DOTAP/DNA. In order to enhance the transfection efficiency, protamine was selected as a DNA-condensing agent to form liposomeprotamineDNA (LPD) ternary complexes. An agarose gel retardation assay was used to examine the DNA binding affinity of the CLs. Following transfection, GFP fluorescence images were captured and flow cytometry was performed to determine the transfection efficiency. Furthermore, an MTT assay was performed to determine the cytotoxicity of the liposome complexes. The final optimal conditions were as follows: 5% glucose as the hydration medium, a molar ratio of DOTAP/chol at 3:1 for the preparation of CLs, a weight ratio of DOTAP/protamine/DNA of 3:0.5:1, with 8 µg plasmid added for the preparation of the LPD complexes. In vitro, the LPD complexes exhibited an enhanced transfection efficiency and low cytotoxicity, which indicated that the presented LPD vector enhanced the transfection efficiency of the CLs. The HepG2 cells were found to have the lowest expression levels of miR145 out of the cell lines tested (A549, BGC-823, HepG2, HeLa, LoVo and MCF-7). Following the transient transfection of the HepG2 cells with miR145, the results revealed that the overexpression of miR145 inhibited the proliferation of the HepG2 cells and downregulated the expression of cyclin-dependent kinase 6 (CDK6), cyclinD1, c-myc, and Sp1 transcription factor (Sp1). In conclusion, in this study, we optimized a liposomebased delivery system for the efficient delivery of miR145 into cancer cells. This may provide a foundation for further research into the use of miR145 in anticancer therapeutics.
Assuntos
Técnicas de Transferência de Genes , Lipossomos , MicroRNAs/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Humanos , Lipossomos/química , Lipossomos/toxicidade , MicroRNAs/administração & dosagem , Nanopartículas , Tamanho da Partícula , Interferência de RNA , TransfecçãoRESUMO
Inflammation is emerging as a new hallmark of cancer. Arachidonic acid (AA) metabolism, the family of cyclooxygenases (COXs) and lipoxygenase (LOX) play important roles in AA-related inflammatory cascades. In 94 colorectal cancer samples collected from the Han population, the immunohistochemical results indicated that 68% of the patients with colorectal cancer had a co-expression of both COX-2 and 5-LOX, while both displayed low expression in the matched normal tissues. In cell lines, three colorectal cancer cell lines exhibited high expression of COX-2 and 5-LOX. During stable silencing of the expression of COX-2 or 5-LOX in LoVo cancer cells, we found that downregulation of either COX-2 or 5-LOX significantly diminished the growth, migration and invasion of the colon cancer cells and specifically, downregulation of COX-2 could elicit upregulation of 5-LOX protein and vice versa. The above results suggested that the simultaneous blocking of COX-2 and 5-LOX activity may bring more potential benefits in managing the progression of colon cancer. Therefore, we sought to explore the effectiveness of a dual COX-2/5-LOX inhibitor darbufelone on the proliferation, migration, invasion and apoptosis of colon cancer cells, as well as the underlying mechanism of action. The results indicated that darbufelone significantly decreased the proliferative and invasive abilities of the colon cancer cells, in a dose-dependent manner. During the study of the related mechanisms, we found an upregulation of p27 and downregulation of cyclin D1 as well as CDK4 after darbufelone treatment, which indicated that darbufelone could arrest the cell cycle of LoVo cells at the G0/G1 phase. Furthermore, the activation of caspase-3 and -9, upregulation of Bax and downregulation of Bcl-2 demonstrated the occurrence of apoptosis by darbufelone. Finally, darbufelone also prevented the migration and invasion of LoVo cells, which may be ascribed to the upregulation of E-cadherin and ZO-1. In summary, our data suggest that the inhibition of both COX-2/5-LOX may be an effective therapeutic approach for colon cancer management, particularly for those patients with high expression of COX-2/5-LOX.
Assuntos
Araquidonato 5-Lipoxigenase/biossíntese , Neoplasias do Colo/tratamento farmacológico , Ciclo-Oxigenase 2/biossíntese , Tiazolidinas/administração & dosagem , Apoptose/efeitos dos fármacos , Araquidonato 5-Lipoxigenase/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica/genética , Proteínas de Neoplasias/biossínteseRESUMO
Vacuole membrane protein 1 (VMP1) was recently found to be involved in the process of tumor metastasis and is also considered to play a vital role in balancing apoptosis and autophagy. In the present study, the expression of VMP1 in colorectal cancer and matched adjacent noncancerous tissues was evaluated by immunohistochemistry (IHC) for studying the role of VMP1 in the process of colorectal cancer. KaplanMeier analysis and the log-rank test were used to calculate the correlation of classic clinicopathological characteristics related to survival and the expression of VMP1. In vitro, a VMP1 stable gene silencing cell model was constructed using a lentiviral vector. The invasive ability and proliferation of colorectal cancer cells were evaluated by Transwell and MTT assays, respectively, and the underlying signaling pathway was explored by western blotting. Additionally, drug susceptibility to cisplatin, oxaliplatin and 5-FU was tested before and after VMP1 knockout. Finally, an animal model was constructed to explore the role of VMP1 in the physiopathologic process of colorectal cancer. Our results indicated that VMP1 showed increased expression in the adjacent non-cancer tissues compared with that in the colorectal cancer tissues. For different stages of colorectal cancer, expression of VMP1 had a negative correlation with the malignancy of the cancer. In clinical research, we also found that the median survival of patients with low VMP1 expression was much shorter than the survival of patients with high expression. In vitro, after infection with the lentivirus, cells with VMP1 knockout gained significant aggressive properties in regards to invasion and proliferation, and the mechanisms may be related to the activation of the PI3K/Akt/ZO-1/E-cadherin pathway. We also found that shVMP1 cells were more sensitive to 5-FU, but not cisplatin and oxaliplatin. Finally, we found a higher number of formed nodules in nude mice after intraperitoneal injection with shVMP1 cells in the in vivo study.