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Glioblastoma (GBM), an aggressive form of brain cancer, has a higher incidence in non-Hispanics when compared to the US Hispanic population. Using data from RT-PCR analysis of 21 GBM tissue from Hispanic patients in Puerto Rico, we identified significant correlations in the gene expression of focal adhesion kinase and proline-rich tyrosine kinase (PTK2 and PTK2B) with NGFR (nerve growth factor receptor), PDGFRB (platelet-derived growth factor receptor B), EGFR (epithelial growth factor receptor), and CXCR1 (C-X-C motif chemokine receptor 1). This study further explores these correlations found in gene expression while accounting for sex and ethnicity. Statistically significant (p < 0.05) correlations with an r value > ±0.7 were subsequently contrasted with mRNA expression data acquired from cBioPortal for 323 GBM specimens. Significant correlations in Puerto Rican male patients were found between PTK2 and PTK2B, NGFR, PDGFRB, EGFR, and CXCR1, which did not arise in non-Hispanic male patient data. The data for Puerto Rican female patients showed correlations in PTK2 with PTK2B, NGFR, PDGFRB, and EGFR, all of which did not appear in the data for non-Hispanic female patients. The data acquired from cBioPortal for non-Puerto Rican Hispanic patients supported the correlations found in the Puerto Rican population for both sexes. Our findings reveal distinct correlations in gene expression patterns, particularly involving PTK2, PTK2B, NGFR, PDGFRB, and EGFR among Puerto Rican Hispanic patients when compared to non-Hispanic counterparts.
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Neoplasias Encefálicas , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Hispânico ou Latino , Transdução de Sinais , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/etnologia , Receptores ErbB/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glioblastoma/genética , Glioblastoma/etnologia , Hispânico ou Latino/genética , Porto Rico , Transdução de Sinais/genéticaRESUMO
Papillary thyroid carcinoma (PTC) presents a significant health concern, particularly among Hispanic women in the United States, who exhibit a disproportionately higher chance of developing an advanced disease when compared to the non-Hispanic population. Emerging evidence suggests coagulation factor X, encoded by the F10 gene, has a potential role in inhibiting cancer cell migration. However, comprehensive investigations into the differential expression patterns of F10 in Hispanic versus non-Hispanic females remain limited. RNA-sequencing data were acquired from the TCGA database for white female patients, 166 non-Hispanic and 25 Hispanic. A statistically significant (p < 0.05) 2.06-fold increase in F10 expression levels was detected in disease-free tumors compared to recurrent PTC tumors. Furthermore, an increase in F10 gene expression levels was also observed, corresponding to approximately a 1.74-fold increase in non-Hispanic patients compared to Hispanic patients. The probability of tumor recurrence was 1.82 times higher in the cohort with low expression of F10 compared to the high-expression cohort, correlating with the lower disease-free rates observed in the Hispanic patient cohort when compared to non-Hispanics. This finding underscores the relevance of ethnic disparities in molecular profiles for understanding cancer susceptibility. Identifying F10 as a potential prognostic biomarker highlights avenues for targeted interventions and contributes to improving diagnostic and treatment strategies for diverse patient populations.
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The majority of glioblastomas (GBMs) recur shortly after tumor resection and recurrent tumors differ significantly from newly diagnosed GBMs, phenotypically and genetically. In this study, using a Gl261-C57Bl/6 mouse glioma implantation model, we identified significant upregulation of proline-rich tyrosine kinase Pyk2 and focal adhesion kinase (FAK) phosphorylation levels-pPyk2 (579/580) and pFAK (925)-without significant modifications in total Pyk2 and FAK protein expression in tumors regrown after surgical resection, compared with primary implanted tumors. Previously, we demonstrated that Pyk2 and FAK are involved in the regulation of tumor cell invasion and proliferation and are associated with reduced overall survival. We hypothesized that the use of inhibitors of Pyk2/FAK in the postsurgical period may reduce the growth of recurrent tumors. Using Western blot analysis and confocal immunofluorescence approaches, we demonstrated upregulation of Cyclin D1 and the Ki67 proliferation index in tumors regrown after resection, compared with primary implanted tumors. Treatment with Pyk2/FAK inhibitor PF-562271, administered through oral gavage at 50 mg/kg daily for two weeks beginning 2 days before tumor resection, reversed Pyk2/FAK signaling upregulation in recurrent tumors, reduced tumor volume, and increased animal survival. In conclusion, the use of Pyk2/FAK inhibitors can contribute to a delay in GBM tumor regrowth after surgical resection.
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Glioblastoma , Glioma , Camundongos , Animais , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Camundongos Endogâmicos C57BL , Quinase 2 de Adesão Focal/genética , Implantação do EmbriãoRESUMO
BACKGROUND: The significant challenge in treating triple-negative breast cancer (TNBC) lies in its high rate of distant metastasis. To address this, inhibiting metastasis formation in TNBC is vital. Rac is a key player in cancer metastasis. Previously, we developed Ehop-016, a Rac inhibitor that successfully reduced tumor growth and metastasis in mice. In this study, we assessed the effectiveness of HV-107, a derivative of Ehop-016, in inhibiting TNBC metastasis at lower doses. METHODS: Rho GTPases activity assays were performed with the use of GST-PAK beads and Rac, Rho, and Cdc42 GLISA. Cell viability was assessed through trypan blue exclusion and MTT assays. Cell cycle analysis was conducted using flow cytometry. To evaluate invading capabilities, transwell assays and invadopodia formation assays were performed. Metastasis formation studies were conducted using a breast cancer xenograft mouse model. RESULTS: HV-107 inhibited Rac activity by 50% in MDA-MB-231 and MDA-MB-468 cells at concentrations of 250-2000 nM, leading to a 90% decrease in invasion and invadopodia activity. Concentrations of 500 nM and above caused dose-dependent reductions in cell viability, resulting in up to 20% cell death after 72 h. Concentrations exceeding 1000 nM upregulated PAK1, PAK2, FAK, Pyk2, Cdc42, and Rho signallings, while Pyk2 was downregulated at 100-500 nM. Through in vitro experiments, optimal concentrations of HV-107 ranging from 250 to 500 nM were identified, effectively inhibiting Rac activity and invasion while minimizing off-target effects. In a breast cancer xenograft model, administration of 5 mg/kg HV-107 (administered intraperitoneally, 5 days a week) reduced Rac activity by 20% in tumors and decreased metastasis by 50% in the lungs and liver. No observed toxicity was noted at the tested doses. CONCLUSION: The findings indicate that HV-107 exhibits promising potential as a therapeutic medication utilizing Rac inhibition mechanisms to address metastasis formation in TNBC.
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Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Quinase 2 de Adesão Focal , Sobrevivência Celular , Citometria de Fluxo , XenoenxertosRESUMO
BACKGROUND: The development of resistance to temozolomide (TMZ), a standard chemotherapeutic, limits the effective treatment of glioblastoma (GBM). Focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (Pyk2) regulate proliferation and invasion of GBM cells. We found that TMZ activates FAK and Pyk2 signaling in GBM. We hypothesized that pharmacological inhibitors of Pyk2/FAK together with TMZ can enhance the inhibitory effect of TMZ on tumor growth and dispersal and improve the treatment outcome. METHODS: Primary human GBM cell cultures and a C57Bl/6-GL261 mouse glioma implantation model were used. Pyk2 (Tyr579/580) and FAK (Tyr925) phosphorylation was analyzed by western blotting. Viability, cell cycle, migration, invasion and invadopodia formation were investigated in vitro. Animal survival, tumor size and invasion, TUNEL apoptotic cell death and the Ki67 proliferation index were evaluated in vivo upon treatment with TMZ (50 mg/kg, once/day, orally) and the Pyk2/FAK inhibitor PF-562271 (once/daily, 50 mg/kg, orally) vs. TMZ monotherapy. RESULTS: In vitro studies revealed significantly reduced viability, cell cycle progression, invasion and invadopodia with TMZ (100 µM) + PF-562271 (16 nM) compared with TMZ alone. In vivo studies demonstrated that combinatorial treatment led to prominent reductions in tumor size and invasive margins, extensive signs of apoptosis and a reduced proliferation index, together with a 15% increase in the survival rate in animals, compared with TMZ monotherapy. CONCLUSION: TMZ + PF-562271 eliminates TMZ-related Pyk2/FAK activation in GBM and improves the treatment efficacy.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Humanos , Camundongos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Quinase 2 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/farmacologia , Glioblastoma/patologia , Glioma/tratamento farmacológico , Glioma/patologia , Camundongos Endogâmicos C57BL , Transdução de Sinais , Temozolomida/uso terapêuticoRESUMO
Glioblastoma is the most aggressive brain cancer and is highly infiltrated with cells of myeloid lineage (TIM) that support tumor growth and invasion. Tumor resection is the primary treatment for glioblastoma; however, the activation state of TIM at the site of tumor resection and its impact on glioma regrowth are poorly understood. Using the C57BL/6/GL261 mouse glioma implantation model, we investigated the state of TIM in the tumor resection area during the post-surgical period. TIM isolated from brain tissue at the resection site were analyzed at 0, 1, 4, 7, 14, and 21 days after tumor resection. An increase in expression of CD86 during the first 7 days after surgical resection and then upregulation of arginase 1 from the 14th to 21st days after resection were detected. Cytokine expression analysis combined with qRT-PCR revealed sustained upregulation of IL4, IL5, IL10, IL12, IL17, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein 1 (MCP1/CCL2) in TIM purified from regrown tumors compared with primary implanted tumors. Flow cytometry analysis revealed increased CD86+/CD206+ population in regrown tumors compared with primary implanted tumors. Overall, we found that TIM in primary implanted tumors and tumors regrown after resection exhibited different phenotypes and cytokine expression patterns.
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Glioblastoma is the most aggressive brain tumor in adults. Multiple lines of evidence suggest that microglia create a microenvironment favoring glioma invasion and proliferation. Our previous studies and literature reports indicated the involvement of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) in glioma cell proliferation and invasion, stimulated by tumor-infiltrating microglia. However, the specific microglia-released factors that modulate Pyk2 and FAK signaling in glioma cells are unknown. In this study, 20 human glioblastoma specimens were evaluated with the use of RT-PCR and western blotting. A Pierson correlation test demonstrated a correlation (0.6-1.0) between the gene expression levels for platelet-derived growth factor ß(PDGFß), stromal-derived factor 1α (SDF-1α), IL-6, IL-8, and epidermal growth factor (EGF) in tumor-purified microglia and levels of p-Pyk2 (Y579/Y580) and p-FAK(Y925) in glioma cells. siRNA knockdown against Pyk2 or FAK in three primary glioblastoma cell lines, developed from the investigated specimens, in combination with the cytokine receptor inhibitors gefitinib (1 µM), DMPQ (200 nM), and burixafor (1 µM) identified EGF, PDGFß, and SDF-1α as key extracellular factors in the Pyk2- and FAK-dependent activation of invadopodia formation and the migration of glioma cells. EGF and IL-6 were identified as regulators of the Pyk2- and FAK-dependent activation of cell viability and mitosis.
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As do many other immunity-related blood cells, platelets release antimicrobial peptides that kill bacteria, fungi, and even certain viruses. Here we review the literature suggesting that there is a similarity between the antimicrobials released by other blood cells and the amyloid-related Aß peptide released by platelets. Analyzing the literature, we also propose that platelet-generated Aß amyloidosis may be more common than currently recognized. This systemic Aß from a platelet source may participate in various forms of amyloidosis in pathologies ranging from brain cancer, glaucoma, skin Aß accumulation, and preeclampsia to Alzheimer's disease and late-stage Parkinson's disease. We also discuss the advantages and disadvantages of specific animal models for studying platelet-related Aß. This field is undergoing rapid change, as it evaluates competing ideas in the light of new experimental observations. We summarized both in order to clarify the role of platelet-generated Aß peptides in amyloidosis-related health disorders, which may be helpful to researchers interested in this growing area of investigation.
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Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/metabolismo , Amiloidose/imunologia , Plaquetas/imunologia , Encéfalo/imunologia , Doença de Parkinson/imunologia , Animais , Autoanticorpos/metabolismo , Modelos Animais de Doenças , HumanosRESUMO
Immunostaining with specific antibodies has shown that innate amyloid beta (Aß) is accumulated naturally in glioma tumors and nearby blood vessels in a mouse model of glioma. In immunofluorescence images, Aß peptide coincides with glioma cells, and enzyme-linked immunosorbent assay (ELISA) have shown that Aß peptide is enriched in the membrane protein fraction of tumor cells. ELISAs have also confirmed that the Aß(1-40) peptide is enriched in glioma tumor areas relative to healthy brain areas. Thioflavin staining revealed that at least some amyloid is present in glioma tumors in aggregated forms. We may suggest that the presence of aggregated amyloid in glioma tumors together with the presence of Aß immunofluorescence coinciding with glioma cells and the nearby vasculature imply that the source of Aß peptides in glioma can be systemic Aß from blood vessels, but this question remains unresolved and needs additional studies.
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Peptídeos beta-Amiloides/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Amyloid beta (Aß) peptides have been implicated in both Alzheimer's disease (AD) and glaucoma and have been shown to be the key etiological factor in these dangerous health complications. On the other hand, it is well known that Aß peptide can be generated from its precursor protein and massively released from the blood to nearby tissue upon the activation of platelets due to their involvement in innate immunity and inflammation processes. Here we review evidence about the development of AD and glaucoma neuronal damage showing their dependence on platelet count and activation. The correlation between the effect on platelet count and the effectiveness of anti-AD and anti-glaucoma therapies suggest that platelets may be an important player in these diseases.
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Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Plaquetas/metabolismo , Glaucoma/sangue , Doença de Alzheimer/etiologia , Glaucoma/etiologia , HumanosRESUMO
In this study, we identified the proton-coupled folate transporter (PCFT) as a route for targeted delivery of drugs to some gliomas. Using the techniques of confocal imaging, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and small interfering (siRNA) knockdown against the PCFT, we demonstrated that Gl261 and A172 glioma cells, but not U87 and primary cultured astrocytes, express the PCFT, which provides selective internalization of folic acid (FA)-conjugated cytochrome c-containing nanoparticles (FA-Cyt c NPs), followed by cell death. The FA-Cyt c NPs (100 µg/mL), had no cytotoxic effects in astrocytes but caused death in glioma cells, according to their level of expression of PCFT. Whole-cell patch clamp recording revealed FA-induced membrane currents in FA-Cyt c NPs-sensitive gliomas, that were reduced by siRNA PCFT knockdown in a similar manner as by application of FA-Cyt c NPs, indicating that the PCFT is a route for internalization of FA-conjugated NPs in these glioma cells. Analysis of human glioblastoma specimens revealed that at least 25% of glioblastomas express elevated level of either PCFT or folate receptor (FOLR1). We conclude that the PCFT provides a mechanism for targeted delivery of drugs to some gliomas as a starting point for the development of efficient methods for treating gliomas with high expression of PCFT and/or FOLR1.
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Neoplasias Encefálicas/metabolismo , Citocromos c/química , Glioma/metabolismo , Nanoconjugados/química , Transportador de Folato Acoplado a Próton/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citocromos c/farmacologia , Ácido Fólico/química , Ácido Fólico/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Nanoconjugados/efeitos adversosRESUMO
Patients infected with human immunodeficiency virus (HIV) are more prone to developing cancers, including glioblastomas (GBMs). The median survival for HIV positive GBM patients is significantly shorter than for those who are uninfected, despite the fact that they receive the same treatments. The nature of the GBMâ»HIV association remains poorly understood. In this study, we analyzed the effect of the HIV envelope glycoprotein gp120 on GBM cell proliferation. Specifically, we performed cell cycle, western blot, protein synthesis and metabolomics analysis as well as ATP production and oxygen consumption assays to evaluate proliferation and metabolic pathways in primary human glioma cell line, U87, A172 cells and in the HIVgp120tg/GL261 mouse model. Glioma cells treated with gp120 (100 ng/mL for 7â»10 days) showed higher proliferation rates and upregulation in the expression of enolase 2, hexokinase and glyceraldehyde-3-phosphate dehydrogenase when compared to untreated cells. Furthermore, we detected an increase in the activity of pyruvate kinase and a higher glycolytic index in gp120 treated cells. Gp120 treated GBM cells also showed heightened lipid and protein synthesis. Overall, we demonstrate that in glioma cells, the HIV envelope glycoprotein promotes proliferation and activation of glycolysis resulting in increased protein and lipid synthesis.
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While it is known that amyloid beta (Aß) deposits are found in different tissues of both Alzheimer's disease (AD) patients and healthy individuals, there remain questions about the physiological role of these deposits, the origin of the Aß peptide, and the mechanisms of its localization to the tissues. Using immunostaining with specific antibodies, as well as enzyme-linked immunosorbent assay, this study demonstrated Aß40 peptide accumulation in the skin during local experimental photothrombosis in mice. Specifically, Aß peptide accumulation was concentrated near the dermal blood vessels in thrombotic skin. It was also studied whether the released peptide affects microorganisms. Application of Aß40 (4 µM) to the external membrane of yeast cells significantly increased membrane conductance with no visible effect on mouse host cells. The results suggest that Aß release in the skin is related to skin injury and thrombosis, and occurs along with clotting whenever skin is damaged. These results support the proposition that Aß release during thrombosis serves as part of a natural defense against infection.
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Peptídeos beta-Amiloides/metabolismo , Pele/metabolismo , Trombose/metabolismo , Animais , Astrócitos/metabolismo , Membrana Celular/metabolismo , Derme/irrigação sanguínea , Feminino , Masculino , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae/metabolismoRESUMO
HIV-1 clades are known to be one of the key factors implicated in modulating HIV-associated neurocognitive disorders. HIV-1 B and C clades account for the majority of HIV-1 infections, clade B being the most neuropathogenic. The mechanisms behind HIV-mediated neuropathogenesis remain the subject of active research. We hypothesized that HIV-1 gp120 clade B and C proteins may exert differential proliferation, cell survival and NeuroAIDS effects in human astrocytoma cells via the Unfolded Protein Response, an endoplasmic reticulum- based cytoprotective mechanism. The differential effect of gp120 clade B and C was evaluated using for the first time a Tandem Mass Tag isobaric labeling quantitative proteomic approach. Flow cytometry analyses were performed for cell cycle and cell death identification. Among the proteins differentiated by HIV-1 gp120 proteins figure cytoskeleton, oxidative stress, UPR markers and numerous glycolytic metabolism enzymes. Our results demonstrate that HIV-1 gp120 B induced migration, proliferative and protective responses granted by the expression of GRP78, while HIV-1 gp120 C induced the expression of key inflammatory and pro-apoptotic markers. These novel findings put forward the first evidence that GRP78 is a key player in HIV-1 clade B and C neuropathogenic discrepancies and can be used as a novel target for immunotherapies.
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The amyloid beta (Aß) peptide and its deposits in the brain are known to be implicated in the neurodegeneration that occurs during Alzheimer's disease (AD). Recently, alternative theories views concerning both the source of this peptide and its functions have been developed. It has been shown that, as in all other known types of amyloidosis, the production of Aß originates in blood cells or cells related to blood plasma, from which it can then spread from the blood to inside the brain, with the greatest concentration around brain blood vessels. In this review, we summarize research progress in this new area and outline some future perspectives. While it is still unclear whether the main source of Aß deposits in AD is the blood, the possibility of blocking the chain of reactions that lead to constant Aß release from the blood to the brain may be exploited in an attempt to reduce the amyloid burden in AD. Solving the problem of Aß accumulation in this way may provide an alternative strategy for developing anti-AD drugs.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Plaquetas/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , HumanosRESUMO
Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.
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Conexina 43/metabolismo , Junções Comunicantes/fisiologia , Neurônios/fisiologia , Poliaminas/administração & dosagem , Acidose , Animais , Cálcio , Linhagem Celular Tumoral , Junções Comunicantes/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Hipercalcemia , Neurônios/metabolismo , Ratos , Espermina/administração & dosagemRESUMO
INTRODUCTION: Platelets contain beta-amyloid precursor protein (APP) as well as Aß peptide (Aß) that can be released upon activation. During thrombosis, platelets are concentrated in clots and activated. METHODS: We used in vivo fluorescent analysis and electron microscopy in mice to determine to what degree platelets are concentrated in clots. We used immunostaining to visualize Aß after photothrombosis in mouse brains. RESULTS: Both in vivo results and electron microscopy revealed that platelets were 300-500 times more concentrated in clots than in non-clotted blood. After thrombosis in control mice, but not in thrombocytopenic animals, Aß immunofluorescence was present inside blood vessels in the visual cortex and around capillaries in the entorhinal cortex. CONCLUSION: The increased concentration of platelets allows enhanced release of Aß during thrombosis, suggesting an additional source of Aß in the brains of Alzheimer's patients that may arise if frequent micro-thrombosis events occur in their brains.
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Peptídeos beta-Amiloides/metabolismo , Plaquetas/metabolismo , Transtornos Cerebrovasculares/metabolismo , Córtex Entorrinal/metabolismo , Trombose/metabolismo , Córtex Visual/metabolismo , Animais , Plaquetas/patologia , Transtornos Cerebrovasculares/patologia , Modelos Animais de Doenças , Córtex Entorrinal/irrigação sanguínea , Córtex Entorrinal/patologia , Feminino , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica , Estimulação Luminosa , Contagem de Plaquetas , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Trombose/patologia , Córtex Visual/irrigação sanguínea , Córtex Visual/patologiaRESUMO
Proteins often possess highly specific biological activities that make them potential therapeutics, but their physical and chemical instabilities during formulation, storage, and delivery have limited their medical use. Therefore, engineering of nanosized vehicles to stabilize protein therapeutics and to allow for targeted treatment of complex diseases, such as cancer, is of considerable interest. A micelle-like nanoparticle (NP) was designed for both, tumor targeting and stimulus-triggered release of the apoptotic protein cytochrome c (Cyt c). This system is composed of a Cyt c NP stabilized by a folate-receptor targeting amphiphilic copolymer (FA-PEG-PLGA) attached to Cyt c through a redox-sensitive bond. FA-PEG-PLGA-S-S-Cyt c NPs exhibited excellent stability under extracellular physiological conditions, whereas once in the intracellular reducing environment, Cyt c was released from the conjugate. Under the same conditions, the folate-decorated NP reduced folate receptor positive HeLa cell viability to 20%, while the same complex without FA only reduced it to 80%. Confocal microscopy showed that the FA-PEG-PLGA-S-S-Cyt c NPs were internalized by HeLa cells and were capable of endosomal escape. The specificity of the folate receptor-mediated internalization was confirmed by the lack of uptake by two folate receptor deficient cell lines: A549 and NIH-3T3. Finally, the potential as antitumor therapy of our folate-decorated Cyt c-based NPs was confirmed with an in vivo brain tumor model. In conclusion, we were able to create a stable, selective, and smart nanosized Cyt c delivery system.
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Citocromos c/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Células A549 , Animais , Apoptose , Citocromos c/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Glioma/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Micelas , Células NIH 3T3 , Polímeros/químicaRESUMO
Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.
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Neoplasias Encefálicas/fisiopatologia , Quinase 2 de Adesão Focal/fisiologia , Glioma/fisiopatologia , Microglia/fisiologia , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/genética , Técnicas de Silenciamento de Genes , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Ratos , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Regulação para CimaRESUMO
Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.