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HIV-associated epidemic Kaposi sarcoma (EpKS) remains one of the most prevalent cancers in sub-Saharan Africa despite the widespread uptake of anti-retroviral therapy and HIV-1 suppression. In an effort to define potential therapeutic targets against KS tumors, we analyzed previously published KS bulk tumor transcriptomics to identify cell surface biomarkers. In addition to upregulated gene expression (>6-fold) in the EpKS tumor microenvironment, biomarkers were selected for correlation with KSHV latency-associated nuclear antigen (LANA) expression. The cell surface glycoprotein genes identified were KDR, FLT4, ADAM12, UNC5A, ZP2, and OX40, as well as the endothelial lineage determinants Prox-1 and CD34. Each protein was evaluated for its expression and co-localization with KSHV LANA using multi-color immunofluorescence in KS tissues, KSHV-infected L1T2 cells, uninfected TIVE cells, and murine L1T2 tumor xenografts. Five surface glycoproteins (KDR, FLT4, UNC5A, ADAM12, and CD34) were associated with LANA-positive cells but were also detected in uninfected cells in the KS microenvironment. In vitro L1T2 cultures showed evidence of only FLT4, KDR, and UNC5A, whereas mouse L1T2 xenografts recapitulated human KS cell surface expression profiles, with the exception of CD34 and Prox-1. In KS tumors, most LANA-positive cells co-expressed markers of vascular as well as lymphatic endothelial lineages, suggesting KS-associated dedifferentiation to a more mesenchymal/progenitor phenotype.
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Proteolysis-targeting chimera (PROTAC) is an attractive technology in drug discovery. Canonically, targets act as a basic starting point in the most previous PROTAC design. Here, we designed degraders considering from the view of clinical benefits. With this novel design, Brigatinib was turned into a degrader SIAIS164018 and endowed with unique features. First, SIAIS164018 could degrade not only ALK fusion proteins in activating or G1202R-mutated form but also mutant EGFR with L858R + T790M, which are two most important targets in non-small-cell lung cancer. Second, SIAIS164018 strongly inhibited cell migration and invasion of Calu-1 and MDA-MB-231. Third and surprisingly, SIAIS164018 degrades several important oncoproteins involved in metastasis such as FAK, PYK2, and PTK6. Interestingly, SIAIS164018 reshuffled the kinome ranking profile when compared to Brigatinib. Finally, SIAIS164018 is orally bioavailable and well tolerated in vivo. SIAIS164018 is an enlightening degrader for us to excavate the charm of protein degradation.
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Quinase do Linfoma Anaplásico/antagonistas & inibidores , Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Quinase do Linfoma Anaplásico/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Humanized bone marrow-liver-thymic (hu-BLT) mice develop a functional immune system in periphery, nevertheless, have a limited reconstitution of human myeloid cells, especially microglia, in CNS. Further, whether bone marrow derived hematopoietic stem and progenitor cells (HSPCs) can enter the brain and differentiate into microglia in adults remains controversial. To close these gaps, in this study we unambiguously demonstrated that human microglia in CNS were extensively reconstituted in adult NOG mice with human interleukin-34 transgene (hIL34 Tg) from circulating CD34+ HSPCs, nonetheless not in hu-BLT NOG mice, providing strong evidence that human CD34+ HSPCs can enter adult brain and differentiate into microglia in CNS in the presence of hIL34. Further, the human microglia in the CNS of hu-BLT-hIL34 NOG mice robustly supported HIV-1 infection reenforcing the notion that microglia are the most important target cells of HIV-1 in CNS and demonstrating its great potential as an in vivo model for studying HIV-1 pathogenesis and evaluating curative therapeutics in both periphery and CNS compartments.
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Modelos Animais de Doenças , Infecções por HIV/virologia , Transplante de Células-Tronco Hematopoéticas/métodos , Interleucinas , Microglia/virologia , Animais , Encéfalo/virologia , Diferenciação Celular , HIV-1 , Humanos , Interleucinas/genética , Camundongos , Camundongos Transgênicos , Microglia/citologia , TransgenesRESUMO
PURPOSE: To compare the use of intraoperative ultrasound with X-ray fluoroscopy during sacral neuromodulation lead electrode placement in patients with neurogenic bladder secondary to spinal cord disease. METHODS: We reviewed the medical records of 52 patients who underwent sacral neuromodulation (SNM) lead electrode implantation under fluoroscopy or ultrasound guidance from July 2016 to July 2019. The operating time, number of electrode contacts with stimulus responses, minimum voltage that causes a stimulus response, and rate of standard lead electrode placement were used to assess the differences between the two methods. All patients were evaluated by recording bladder diaries, postvoid residual volumes before and during the testing period. Permanent SNM implantation is acceptable if symptoms improve by at least 50%. RESULTS: The operating time decreased from 87.1 ± 25.19 min in the X-ray group to 68.2 ± 25.20 min (p < 0.05) in the ultrasound group. The number of electrode contacts with stimulus responses, rate of standard lead electrode placement, and implantable pulse generator (IPG) placement rate were not significantly different between the two groups (p > 0.05). There was no radiation exposure during the operation in the ultrasound group. No incisional infections, hematomas, or other critical complications were reported in either groups. CONCLUSION: Ultrasound can be applied to safely place lead electrode for sacral neuromodulation and leads to no radiation exposure to the patient, surgeon, and operating room staff and a shortened operating time while maintaining the same efficacy as X-ray.
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Terapia por Estimulação Elétrica/métodos , Eletrodos Implantados , Implantação de Prótese , Bexiga Urinaria Neurogênica/terapia , Adulto , Idoso , Feminino , Fluoroscopia , Humanos , Período Intraoperatório , Plexo Lombossacral , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Doenças da Medula Espinal/complicações , Cirurgia Assistida por Computador , Ultrassonografia , Bexiga Urinaria Neurogênica/etiologiaRESUMO
Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone throughout life, where they can be activated in response to physiological and pathophysiological stimuli. A recent study indicates metabotropic glutamate receptor 4 (mGluR4) is involved in regulating NSPCs behaviors. Therefore, defining mGluR4 function in NSPCs is necessary for determining novel strategies to enhance the intrinsic potential for brain regeneration after injuries. In this study, mGluR4 was functionally expressed in SVZ-derived NSPCs from male Sprague-Dawley rats, in which the cyclic adenosine monophosphate concentration was reduced after treatment with the mGluR4-specific agonist VU0155041. Additionally, lateral ventricle injection of VU0155041 significantly decreased 5-bromo-2'-deoxyuridine (BrdU)+ and Ki67+ cells, while increased Doublecortin (DCX)/BrdU double-positive cells in SVZ. In cultured NSPCs, mGluR4 activation decreased the ratio of BrdU+ cells, G2/M-phase cells, and inhibited Cyclin D1 expression, whereas it increased neuron-specific class III ß-tubulin (Tuj1) expression and the number of Tuj1, DCX, and PSA-NCAM-positive cells. However, pharmacological blocking mGluR4 with the antagonist MSOP or knockdown of mGluR4 abolished the effects of VU0155041 on NSPCs proliferation and neuronal differentiation. Further investigation demonstrated that VU0155041 treatment down-regulated AKT phosphorylation and up-regulated expression of the phosphatase and tensin homolog protein (PTEN) in NSPCs culture. Moreover VU0155041-induced proliferating inhibition and neuronal differentiating amplification in NSPCs were significantly hampered by VO-OHpic, a PTEN inhibitor. We conclude that activation of mGluR4 in SVZ-derived NSPCs suppresses proliferation and enhances their neuronal differentiation, and regulation of PTEN may be involved as a potential intracellular target of mGluR4 signal. Cover Image for this issue: https://doi.org/10.1111/jnc.15052.
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Diferenciação Celular/fisiologia , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/metabolismo , PTEN Fosfo-Hidrolase/biossíntese , Receptores de Glutamato Metabotrópico/metabolismo , Anilidas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Ácidos Cicloexanocarboxílicos/farmacologia , Relação Dose-Resposta a Droga , Proteína Duplacortina , Expressão Gênica , Ventrículos Laterais/citologia , Ventrículos Laterais/efeitos dos fármacos , Masculino , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , PTEN Fosfo-Hidrolase/genética , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistasRESUMO
EML4-ALK and NPM-ALK fusion proteins possess constitutively activated ALK (anaplastic lymphoma kinase) activity, which in turn leads to the development of non-small cell lung cancer and anaplastic large-cell lymphomas (ALCLs). FDA-approved ALK inhibitor drugs cause significant cancer regression. However, drug resistance eventually occurs and it becomes a big obstacle in clinic. Novel proteolysis targeting chimera (PROTAC) technology platform provides a potential therapeutic strategy for drug resistance. Herein, we designed and synthesized a series of ALK PROTACs based on Brigatinib and VHL-1 conjunction, and screened SIAIS117 as the best degrader which not only blocked the growth of SR and H2228 cancer cell lines, but also degraded ALK protein. In addition, SIAIS117 also showed much better growth inhibition effect than Brigatinib on 293T cell line that exogenously expressed G1202R-resistant ALK proteins. Furthermore, it also degraded G1202R mutant ALK protein in vitro. At last, it has the potentially anti-proliferation ability of small cell lung cancer. Thus, we have successfully generated the degrader SIAIS117 that can potentially overcome resistance in cancer targeted therapy.
Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Desenvolvimento de Medicamentos , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinase do Linfoma Anaplásico/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
The activation of microglia in response to intracerebral hemorrhagic stroke is one of the principal components of the progression of this disease. It results in the formation of pro-inflammatory cytokines that lead to neuronal death, a structural deterioration that, in turn interferes with functional recovery. Metabotropic glutamate receptor 5 (mGluR5) is highly expressed in reactive microglia and is involved in the pathological processes of brain disorders, but its role in intracerebral hemorrhage (ICH) remains unknown. We hypothesized that mGluR5 regulates microglial activation and ICH maintenance. In this study, collagenase-induced ICH mice received a single intraperitoneal injection of the mGluR5 antagonist-, MTEP, or vehicle 2 h after injury. We found that acute ICH upregulated mGluR5 and microglial activation. mGluR5 was highly localized in reactive microglia in the peri-hematomal cortex and striatum on days 3 and 7 post-ICH. The MTEP-mediated pharmacological inhibition of mGluR5 in vivo resulted in the substantial attenuation of acute microglial activation and IL-6, and TNF-α release. We also showed that the blockade of mGluR5 markedly reduced cell apoptosis, and neurodegeneration and markedly elevated neuroprotection. Furthermore, the MTEP-mediated inhibition of mGluR5 significantly reduced the lesion volume and improved functional recovery. Taken together, our results demonstrate that ICH injury enhances mGluR5 expression in the acute and subacute stages and that mGluR5 is highly localized in reactive microglia. The blockade of mGluR5 reduces ICH-induced acute microglial activation, provides neuroprotection and promotes neurofunctional recovery after ICH. The inhibition of mGluR5 may be a relevant therapeutic target for intracerebral hemorrhagic stroke.
Assuntos
Hemorragia Cerebral/prevenção & controle , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Piridinas/uso terapêutico , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Tiazóis/uso terapêutico , Animais , Morte Celular , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patologia , Masculino , Camundongos , Microglia/metabolismo , Microglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Piridinas/farmacologia , Distribuição Aleatória , Receptor de Glutamato Metabotrópico 5/metabolismo , Tiazóis/farmacologiaRESUMO
We previously showed that rhesus macaques neonatally infected with simian immunodeficiency virus (SIV) do not develop SIV encephalitis (SIVE) and maintain low brain viral loads despite having similar plasma viral loads compared to SIV-infected adults. We hypothesize that differences in myeloid cell populations that are the known target of SIV and HIV in the brain contribute to the lack of neonatal susceptibility to lentivirus-induced encephalitis. Using immunohistochemistry and immunofluorescence microscopy, we examined the frontal cortices from uninfected and SIV-infected infant and adult macaques (n = 8/ea) as well as adults with SIVE (n = 4) to determine differences in myeloid cell populations. The number of CD206+ brain perivascular macrophages (PVMs) was significantly greater in uninfected infants than in uninfected adults and was markedly lower in SIV-infected infants while microglia numbers were unchanged across groups. CD206+ PVMs, which proliferate after infection in SIV-infected adults, did not undergo proliferation in infants. While virtually all CD206+ cells in adults are also CD163+, infants have a distinct CD206 single-positive population in addition to the double-positive population commonly seen in adults. Notably, we found that more than 60% of these unique CD206+CD163- PVMs in SIV-infected infants were positive for cleaved caspase-3, an indicator of apoptosis, and that nearly 100% of this subset were concomitantly positive for the necroptosis marker receptor-interacting protein kinase-3 (RIP3). These findings show that distinct subpopulations of PVMs found in infants undergo programmed cell death instead of proliferation following SIV infection, which may lead to the absence of PVM-dependent SIVE and the limited size of the virus reservoir in the infant brain.
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Encéfalo/patologia , Macrófagos/patologia , Necroptose/fisiologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Animais , Feminino , Macaca mulatta , Masculino , Vírus da Imunodeficiência SímiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Natural hosts of simian immunodeficiency virus (SIV) avoid AIDS despite lifelong infection. Here, we examined how this outcome is achieved by comparing a natural SIV host, African green monkey (AGM) to an AIDS susceptible species, rhesus macaque (RM). To asses gene expression profiles from acutely SIV infected AGMs and RMs, we developed a systems biology approach termed Conserved Gene Signature Analysis (CGSA), which compared RNA sequencing data from rectal AGM and RM tissues to various other species. We found that AGMs rapidly activate, and then maintain, evolutionarily conserved regenerative wound healing mechanisms in mucosal tissue. The wound healing protein fibronectin shows distinct tissue distribution and abundance kinetics in AGMs. Furthermore, AGM monocytes exhibit an embryonic development and repair/regeneration signature featuring TGF-ß and concomitant reduced expression of inflammatory genes compared to RMs. This regenerative wound healing process likely preserves mucosal integrity and prevents inflammatory insults that underlie immune exhaustion in RMs.
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Fibronectinas/imunologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Fator de Crescimento Transformador beta/imunologia , Cicatrização/imunologia , Animais , Chlorocebus aethiops/genética , Chlorocebus aethiops/imunologia , Progressão da Doença , Fibronectinas/metabolismo , Mucosa Intestinal/metabolismo , Macaca mulatta/genética , Macaca mulatta/imunologia , Macrófagos/metabolismo , Reto/imunologia , Reto/metabolismo , Vírus da Imunodeficiência Símia , Biologia de Sistemas , Transcriptoma , Fator de Crescimento Transformador beta/genética , Cicatrização/genéticaRESUMO
Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.
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Microbioma Gastrointestinal , Sistema Imunitário , Modelos Animais , Animais , Transplante de Microbiota Fecal , Humanos , Camundongos , Camundongos Endogâmicos NOD , RNA Ribossômico 16SRESUMO
Tripartite motif 32 (TRIM32) is a member of TRIM family that plays a potential role in neural regeneration. However, the biological function of TRIM32 in cerebral ischemia reperfusion injury has not been investigated. In the present study, we evaluated the expression level of TRIM32 in hippocampal neurons following oxygen-glucose deprivation/reperfusion (OGD/R). The results showed that TRIM32 expression was significantly elevated in hippocampal neurons subjected to OGD/R as compared to the neurons cultured in the normoxia condition. To further evaluate the role of TRIM32, hippocampal neurons were transfected with TRIM32 small interfering RNA (si-TRIM32) to knock down TRIM32. We found that knockdown of TRIM32 improved cell viability of OGD/R-stimulated hippocampal neurons. Generation of reactive oxygen species was decreased, while contents of superoxide dismutase and glutathione peroxidase were increased after si-TRIM32 transfection. Knockdown of TRIM32 suppressed cell apoptosis, as proved by the increased bcl-2 expression along with decreased bax expression and caspase-3 activity. We also found that TRIM32 knockdown enhanced OGD/R-induced activation of Nrf2 signaling pathway in hippocampal neurons. Furthermore, siRNA-Nrf2 was transfected to knock down Nrf2. SiRNA-Nrf2 transfection reversed the protective effects of TRIM32 knockdown on neurons. These data suggested that knockdown of TRIM32 protected hippocampal neurons from OGD/R-induced oxidative injury through activating Nrf2 signaling pathway.
Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neuroproteção/fisiologia , Oxigênio/metabolismo , Gravidez , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Angiogenesis is induced immediately after cerebral ischemia and plays a pivotal role in the strategy against ischemic injury. We hypothesized that the coordinated interaction between microvessels and neurons was altered immediately after stroke, and microvessels and neurons would show the temporal specificity of angiogenic gene profiles after cerebral ischemia. Microvessels and neurons were harvested in the ischemic penumbra of rat brain using the PixCell II laser capture microdissection (LCM) instrument. After RNA isolation, T7 and gene-specific primer RNA linear amplification were performed, and angiogenic functional grouping cDNA profiling was analyzed in LCM samples. cDNA microarray results showed there were 35 (36.46%) and 27 (28.13%) genes expression changes in the microvessels, while 25 (26.04%) and 31 (32.29%) genes were changed in the neurons at 2 h and 24 h after cerebral ischemia. Members of growth factors and receptors, cytokines and chemokines, adhesion molecules, matrix proteins, proteases, and inhibitors showed temporal and spatial differentiation in the microvessels and neurons after cerebral ischemia. This finding will help to understand the coordination and interaction between microvessels and neurons, and to elucidate the molecular mechanisms of angiogenesis after brain ischemic injury.
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Infarto da Artéria Cerebral Média/metabolismo , Microvasos/metabolismo , Neovascularização Fisiológica/genética , Neurônios/metabolismo , Transcriptoma , Animais , Perfilação da Expressão Gênica , Infarto da Artéria Cerebral Média/patologia , Microdissecção e Captura a Laser , Microvasos/patologia , Neurônios/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Endoplasmic reticulum (ER) stress caused by ß-amyloid protein (Aß) may play an important role in the pathogenesis of Alzheimer disease (AD). Our previous data have indicated that tanshinone IIA (tan IIA) protected primary neurons from Aß induced neurotoxicity. To further explore the neuroprotection of tan IIA, here we study the effects of tan IIA on the ER stress response in oligomeric Aß1-42 (oAß1-42)-induced SH-SY5Y cell injury. Our data showed that tan IIA pretreatment could increase cell viability and inhibit apoptosis caused by oAß1-42. Furthermore, tan IIA markedly suppressed ER dilation and prevented oAß1-42-induced abnormal expression of glucose regulated protein 78 (GRP78), initiation factor 2α (eIF2α), activating transcription factor 6 (ATF6), as well as inhibited the activation of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) pathways. Moreover, tan IIA ameliorated oAß1-42-induced Bcl-2/Bax ratio reduction, prevented cytochrome c translocation into cytosol from mitochondria, reduced oAß1-42-induced cleavage of caspase-9 and caspase-3, suppressed caspase-3/7 activity, and increased mitochondrial membrane potential (MMP) and ATP content. Meanwhile, oAß1-42-induced cell apoptosis and activation of ER stress can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA). Taken together, these data indicated that tan IIA protects SH-SY5Y cells against oAß1-42-induced apoptosis through attenuating ER stress, modulating CHOP and JNK pathways, decreasing the expression of cytochrome c, cleaved caspase-9 and cleaved caspase-3, as well as increasing the ratio of Bcl-2/Bax, MMP and ATP content. Our results strongly suggested that tan IIA may be effective in treating AD associated with ER stress.
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Abietanos/farmacologia , Peptídeos beta-Amiloides/toxicidade , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
BACKGROUND: MicroRNAs play crucial roles in tumorigenesis and tumor progression. miR-770 has been reported to be downregulated in several cancers and affects cancer cell proliferation, apoptosis, metastasis and drug resistance. However, the role and underlying molecular mechanism of miR-770 in human glioma remain unknown and need to be further elucidated. METHODS: The expression of miR-770 in glioma tissues and cell lines was measured by quantitative real-time PCR (qRT-PCR) to explore the association of miR-770 expression with clinicopathological characteristics. The expression of CDK8 was detected by qRT-PCR and Western blotting in glioma tissues. A target prediction program and a dual-luciferase reporter assay were used to confirm that CDK8 is a target gene of miR-770. MTT and cell counting assays were used to assess the effect of miR-770 on glioma cell proliferation. The cell cycle distribution and apoptosis were examined by flow cytometry. CDK8 siRNA and overexpression were used to further confirm the function of the target gene. RESULTS: We demonstrated that miR-770 expression was downregulated in human glioma tissues and cell lines. The overexpression of miR-770 inhibited glioma cell proliferation and cell cycle G1-S transition and induced apoptosis. The inhibition of miR-770 facilitated cell proliferation and G1-S transition and suppressed apoptosis. miR-770 expression was inversely correlated with CDK8 expression in glioma tissues. CDK8 was confirmed to be a direct target of miR-770 by using a luciferase reporter assay. The overexpression of miR-770 decreased CDK8 expression at both the mRNA and protein levels, and the suppression of miR-770 increased CDK8 expression. Importantly, CDK8 silencing recapitulated the cellular and molecular effects observed upon miR-770 overexpression, and CDK8 overexpression eliminated the effects of miR-770 overexpression on glioma cells. Moreover, both exogenous expression of miR-770 and silencing of CDK8 resulted in suppression of the Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our study demonstrates that miR-770 inhibits glioma cell proliferation and G1-S transition and induces apoptosis through suppression of the Wnt/ß-catenin signaling pathway by targeting CDK8. These findings suggest that miR-770 plays a significant role in glioma progression and serves as a potential therapeutic target for glioma.
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Brain microvascular endothelial cells (BMECs) play vital roles in cerebral ischemia, during which many signal pathways mediate BMECs apoptosis. In this study, we explored the potential role of Wnt3α/ß-catenin signal in BMECs apoptosis induced by ischemia. Here, we found that oxygen-glucose deprivation (OGD) could induce apoptosis of BMECs with Wnt3a mRNA expression decrease. Meanwhile, activation Wnt3a/ß-catenin signal with exogenous Wnt3α protein (100 ng/ml) or Lithium Chloride (LiCl, 4 mM) decreased significantly apoptosis of BMECs induced by OGD with increasing expression of Bcl-2 in the whole cell and ß-catenin in the nucleus. But, inhibition Wnt3a/ß-catenin signal with DKK1 (100 ng/ml) or 2.4-diamino quinazoline (DQ, 0.2 µM) increased apoptosis of BMECs with decreasing expression of Bcl-2. These results suggest that activation Wnt3α/ß-catenin signal attenuate apoptosis of BMECs induced by ischemia.
Assuntos
Apoptose/efeitos dos fármacos , Cérebro/irrigação sanguínea , Células Endoteliais/efeitos dos fármacos , Glucose/deficiência , Oxigênio/metabolismo , Proteína Wnt3/metabolismo , beta Catenina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Circulação Cerebrovascular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glucose/metabolismo , Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cloreto de Lítio/farmacologia , Microvasos/citologia , Quinazolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3/agonistas , beta Catenina/agonistasRESUMO
Abnormal hippocampal neurogenesis after acute seizures has been well addressed. However, whether newly generated cells continued to be disturbed even they were born in the chronic stage after pilocarpine-induce status epilepticus has remained elusive. Labeling dividing progenitors and their progeny with retroviral vector expressing green fluorescent protein or proliferation marker 5-bromo-2'-deoxyuridine at 3 months post pilocarpine-induced status epilepticus in mice, a spot of newly born neurons exhibiting hilar ectopic location (4.57±2.3%), aberrant basal dendrites (8.09±1.5%) and abnormal axon sprouting into inner molecular layer of dentate gyrus was identified when examined 6 weeks later. No significant difference on the amount of mossy fiber sprouting was found when cohorts of newborn cells were eliminated by methylazoxymethanol acetate injection initiated at 3 months after SE, suggesting that adult generated neurons in the chronically epileptic hippocampus don't contribute a lot to the mossy fiber sprouting. These results indicated that the aberrant neurogenesis in the chronically epileptic hippocampus occurs only in a small population of newly generated granule cells.
Assuntos
Epilepsia/fisiopatologia , Hipocampo/fisiopatologia , Neurogênese/fisiologia , Animais , Bromodesoxiuridina , Movimento Celular , Doença Crônica , Modelos Animais de Doenças , Epilepsia/patologia , Feminino , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/patologia , Camundongos , Neurônios/patologia , Neurônios/fisiologia , PilocarpinaRESUMO
MicroRNA-25 (miR-25) has been reported to be a major miRNA marker in neural cells and is strongly expressed in ischemic brain tissues. However, the precise mechanism and effect of miR-25 in cerebral ischemia/reperfusion (I/R) injury needs further investigations. In the present study, the oxygen-glucose deprivation (OGD) model was constructed in human SH-SY5Y and IMR-32 cells to mimic I/R injury and to evaluate the role of miR-25 in regulating OGD/reperfusion (OGDR)-induced cell apoptosis. We found that miR-25 was downregulated in the OGDR model. Overexpression of miR-25 via miRNA-mimics transfection remarkably inhibited OGDR-induced cell apoptosis. Moreover, Fas was predicted as a target gene of miR-25 through bioinformatic analysis. The interaction between miR-25 and 3'-untranslated region (UTR) of Fas mRNA was confirmed by dual-luciferase reporter assay. Fas protein expression was downregulated by miR-25 overexpression in OGDR model. Subsequently, the small interfering RNA (siRNA)-mediated knockdown of Fas expression also inhibited cell apoptosis induced by OGDR model; in contrast, Fas overexpression abrogated the protective effects of miR-25 on OGDR-induced cells. Taken together, our results indicate that the upregulation of miR-25 inhibits cerebral I/R injury-induced apoptosis through downregulating Fas/FasL, which will provide a promising therapeutic target.
Assuntos
Apoptose , Glucose/deficiência , MicroRNAs/genética , Neurônios/metabolismo , Oxigênio/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Regulação para Baixo , Proteína Ligante Fas/metabolismo , Humanos , MicroRNAs/metabolismo , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Emerging evidence has linked chronic temporal lobe epilepsy to dramatically reduced neurogenesis in the dentate gyrus. However, the profile of different components of neurogenesis in the chronically epileptic hippocampus is still unclear, especially the incorporation of newly generated cells. To address the issue, newly generated cells in the sub-granular zone of the dentate gyrus were labeled by the proliferation marker bromodeoxyuridine (BrdU) or retroviral vector expressing green fluorescent protein 2 months after pilocarpine-induced status epilepticus. The newly generated neurons that extended axons to CA3 area or integrated into memory circuits were visualized by cholera toxin B subunit retrograde tracing, and detecting activation of BrdU(+) cells following a recall of spatial memory test at the chronic stage of TLE. We found that the microenvironment was still able to sustain significant neuronal differentiation of newly generated cells at 2 months post-status epilepticus time-point, and newly added neurons into granular cell layer were still able to integrate into neuronal circuitry, both anatomically and functionally. Quantified analyses of BrdU(+) or Ki-67(+) cells demonstrated that there was a reduced proliferation of progenitor cells and diminished survival of newly generated cells in the epileptic hippocampus. Both decreased levels of neurotrophic factors in the surrounding milieu and cell loss in the CA3 area might contribute the decreased production of new cells and their survival following chronic epilepsy. These results suggest that decreased neurogenesis in the chronically epileptic hippocampus 2 months post status epilepticus is not associated with altered integration of newly generated neurons, and that developing strategies to augment hippocampal neurogenesis in chronic epilepsy might be protective.
Assuntos
Hipocampo/patologia , Hipocampo/fisiopatologia , Vias Neurais/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Estado Epiléptico/patologia , Animais , Diferenciação Celular , Movimento Celular , Toxina da Cólera/metabolismo , Modelos Animais de Doenças , Feminino , Seguimentos , Camundongos , Camundongos Endogâmicos , Agonistas Muscarínicos , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Pilocarpina/toxicidade , Retroviridae/genética , Estado Epiléptico/induzido quimicamente , Células-Tronco , Fatores de Tempo , Transdução GenéticaRESUMO
Promoting both endogenous and exogenous neural stem cells' (NSCs) survival in the hostile host environments is essential to cell replacement therapy for central nervous system (CNS) disorders. Type 4 metabotropic glutamate receptor (mGluR4), one of the members of mGluRs, has been shown to protect neurons from acute and chronic excitotoxic insults in various brain damages. The present study investigated the preventive effects of mGluR4 on NSC injury induced by oxidative stress. Under challenge with H2O2, loss of cell viability was observed in cultured rat NSCs, and treatment with selective mGluR4 agonist VU0155041 conferred protective effects against the loss of cellular viability in a concentration-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Pretreatment of VU0155041 (30 µM) also inhibited the excessive NSC death induced by H2O2, and group III mGluRs antagonist (RS)-a-methylserine-O-phosphate (MSOP) or gene-targeted knockdown abolished the protective action of mGluR4, indicated by propidium iodide-Hoechst and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining. Western blot assay demonstrated that mGluR4 activation reversed the decreased procaspase-8/9/3and the destructed Bcl-2/Bax expressing balance, and likewise, MSOP and mGluR4 knockdown abrogated the action of mGluR4 activity. Furthermore, inhibition of JNK and p38 mitogen-activated protein kinases (MAPKs) were observed after mGluR4 activation, and as paralleling control, JNK-specific inhibitor SP600125 and p38-specific inhibitor SB203580 significantly rescued the H2O2-mediated NSC apoptosis and cleavage of procaspase-3. We suggest that activation of mGluR4 prevents oxidative stress-induced NSC death and apoptotic-associated protein activities with involvement of inhibiting the JNK and p38 pathways in cell culture. Our findings may help to develop strategies for enhancing the resided and transplanted NSC survival after oxidative stress insult of CNS.