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Bevacizumab, a VEGF-targeting monoclonal antibody, may trigger an infiltrative growth pattern in glioblastoma. We investigated this pattern using both a human specimen and rat models. In the human specimen, a substantial fraction of infiltrating tumor cells were located along perivascular spaces in close relationship with endothelial cells. Brain xenografts of U87MG cells treated with bevacizumab were smaller than controls (p = 0.0055; Student t-test), however, bands of tumor cells spread through the brain farther than controls (p < 0.001; Student t-test). Infiltrating tumor Cells exhibited tropism for vascular structures and propensity to form tubules and niches with endothelial cells. Molecularly, bevacizumab triggered an epithelial to mesenchymal transition with over-expression of the receptor Plexin Domain Containing 1 (PLXDC1). These results were validated using brain xenografts of patient-derived glioma stem-like cells. Enforced expression of PLXDC1 in U87MG cells promoted brain infiltration along perivascular spaces. Importantly, PLXDC1 inhibition prevented perivascular infiltration and significantly increased the survival of bevacizumab-treated rats. Our study indicates that bevacizumab-induced brain infiltration is driven by vascular endothelium and depends on PLXDC1 activation of tumor cells.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Bevacizumab/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Endotélio/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Animais , Antineoplásicos Imunológicos/uso terapêutico , Bevacizumab/uso terapêutico , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais , Endotélio/citologia , Endotélio/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Nus , Receptores de Superfície Celular/genética , Análise de Sobrevida , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We hypothesized that in glioblastoma recurring after radiotherapy, a condition whereby the brain endothelium undergoes radiation-induced senescence, tumor cells with endothelial phenotype may be relevant for tumor neovascularization. Matched glioblastoma samples obtained at primary surgery and at surgery for tumor recurrence after radiotherapy, all expressing epidermal growth factor receptor variant III (EGFRvIII), were assessed by a technique that combines fluorescent in situ hybridization (FISH) for the EGFR/CEP7 chromosomal probe with immunostaining for endothelial cells (CD31) and activated pericytes (α Smooth Muscle Actin). Five EGFRvIII-expressing paired primary/recurrent glioblastoma samples, in which the tumor cells showed EGFR/CEP7 amplification, were then assessed by CD31 and α Smooth Muscle Actin immunofluorescence. In glomeruloid bodies, the ratio between CD31+ cells with amplified EGFR/CEP7 signal and the total CD31+ cells was 0.23 ± 0.09 (mean ± sem) and 0.63 ± 0.07 in primary tumors and in recurrent ones, respectively (p < 0.002, Student-t test). In capillaries, the ratio of CD31+ cells with amplified EGFR/CEP7 over the total CD31+ cells lining the capillary lumen was 0.21 ± 0.06 (mean ± sem) and 0.42 ± 0.07 at primary surgery and at recurrence, respectively (p < 0.005, Student-t test). Expression of α Smooth Muscle Actin by cells with EGFR/CEP7 amplification was not observed. Then, in glioblastoma recurring after radiotherapy, where the brain endothelium suffers from radiation-induced cell senescence, tumor-derived endothelium plays a role in neo-vascularization.
Assuntos
Neoplasias Encefálicas/patologia , Transdiferenciação Celular/fisiologia , Células Endoteliais/patologia , Glioblastoma/patologia , Recidiva Local de Neoplasia/patologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/radioterapia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Hibridização in Situ Fluorescente , Camundongos , Recidiva Local de Neoplasia/genéticaRESUMO
In the neocortex, the coexistence of temporally locked excitation and inhibition governs complex network activity underlying cognitive functions, and is believed to be altered in several brain diseases. Here we show that this equilibrium can be unlocked by increased activity of layer 5 pyramidal neurons of the mouse neocortex. Somatic depolarization or short bursts of action potentials of layer 5 pyramidal neurons induced a selective long-term potentiation of GABAergic synapses (LTPi) without affecting glutamatergic inputs. Remarkably, LTPi was selective for perisomatic inhibition from parvalbumin basket cells, leaving dendritic inhibition intact. It relied on retrograde signaling of nitric oxide, which persistently altered presynaptic GABA release and diffused to inhibitory synapses impinging on adjacent pyramidal neurons. LTPi reduced the time window of synaptic summation and increased the temporal precision of spike generation. Thus, increases in single cortical pyramidal neuron activity can induce an interneuron-selective GABAergic plasticity effectively altering the computation of temporally coded information.
Assuntos
Células Piramidais/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo L/fisiologia , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Plasticidade Neuronal/fisiologia , Neurônios , Técnicas de Patch-Clamp , Ácido gama-Aminobutírico/fisiologiaRESUMO
SNAP-25 is a key component of the synaptic-vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP-25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP-25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP-25 levels at 13-14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage-gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13-14 DIV neurons with reduced SNAP-25 expression show paired-pulse depression as opposed to paired-pulse facilitation occurring in their wild-type counterparts. This phenotype disappears with synapse maturation. As alterations in short-term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP-25.
Assuntos
Ácido Glutâmico/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/fisiologia , Proteína 25 Associada a Sinaptossoma/antagonistas & inibidores , Proteína 25 Associada a Sinaptossoma/genética , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence is used by neurosurgeons to identify the tumor cells of high-grade gliomas during operation. However, the issue of whether 5-ALA-induced PpIX fluorescence consistently stains all the tumor cells is still debated. Here, we assessed the cytoplasmatic signal of 5-ALA by fluorescence microscopy in a series of human gliomas. As tumor markers, we used antibodies against collapsin response-mediated protein 5 (CRMP5), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and anti-isocitrate dehydrogenase 1 (IDH1). In grade III-IV gliomas, the signal induced by 5-ALA was detected in 32.7-75.5 percent of CRMP5-expressing tumor cells. In low-grade gliomas (WHO grade II), the CRMP5-expressing tumor cells did not fluoresce following 5-ALA. Immunofluorescence with antibodies that stain various components of the blood-brain barrier (BBB) suggested that 5-ALA does not cross the un-breached BBB, in spite of its small dimension. To conclude, 5-ALA-induced PpIX fluorescence has an established role in high-grade glioma surgery, but it has limited usefulness in surgery for low-grade glioma, especially when the BBB is preserved.
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Adult neurogenesis in the dentate gyrus plays a critical role in hippocampus-dependent spatial learning. It remains unknown, however, how new neurons become functionally integrated into spatial circuits and contribute to hippocampus-mediated forms of learning and memory. To investigate these issues, we used a mouse model in which the differentiation of adult-generated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. In contrast to previous studies that affected the number of newly generated neurons, this strategy selectively changes their timing of differentiation. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation and significantly reduced dendritic arborization and spine density. Functionally, this genetic manipulation specifically affected different hippocampus-dependent learning and memory tasks, including contextual fear conditioning, and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows, revealed that the new, adult-generated neurons up to 3-4 weeks of age are required not only to acquire new spatial information but also to use previously consolidated memories. Thus, the correct unwinding of these key memory functions, which can be an expression of the ability of adult-generated neurons to link subsequent events in memory circuits, is critically dependent on the correct timing of the initial stages of neuron maturation and connection to existing circuits.
Assuntos
Diferenciação Celular/fisiologia , Hipocampo/citologia , Memória , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Animais , Genes Supressores de Tumor , Hipocampo/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/fisiologia , Percepção Espacial/fisiologia , Fatores de Tempo , Proteínas Supressoras de TumorRESUMO
Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs' fundamental biology is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit elevated levels of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components is sufficient to induce ciliogenesis in a subset of GSCs via relocating platelet-derived growth factor receptor-alpha (PDGFR-α) to a newly induced cilium. Importantly, restoring ciliogenesis enabled GSCs to switch from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a role for cilium as a molecular switch in determining GSCs' fate and suggest cilium induction as an attractive strategy to intervene in GSCs proliferation.
Assuntos
Neoplasias Encefálicas/patologia , Diferenciação Celular/fisiologia , Glioma/patologia , Recidiva Local de Neoplasia/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Autorrenovação Celular/fisiologia , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismoRESUMO
Abnormalities in arterial versus venous endothelial cell identity and dysregulation of angiogenesis are deemed important in the pathophysiology of brain arteriovenous malformations (AVMs). The Sonic hedgehog (Shh) pathway is crucial for both angiogenesis and arterial versus venous differentiation of endothelial cells, through its dual role on the vascular endothelial growth factor/Notch signaling and the nuclear orphan receptor COUP-TFII. In this study, we show that Shh, Gli1 (the main transcription factor of the Shh pathway), and COUP-TFII (a target of the non-canonical Shh pathway) are aberrantly expressed in human brain AVMs. We also show that implantation of pellets containing Shh in the cornea of Efnb2/LacZ mice induces growth of distinct arteries and veins, interconnected by complex sets of arteriovenous shunts, without an interposed capillary bed, as seen in AVMs. We also demonstrate that injection in the rat brain of a plasmid containing the human Shh gene induces the growth of tangles of tortuous and dilated vessels, in part positive and in part negative for the arterial marker αSMA, with direct connections between αSMA-positive and -negative vessels. In summary, we show that the Shh pathway is active in human brain AVMs and that Shh-induced angiogenesis has characteristics reminiscent of those seen in AVMs in humans.
Assuntos
Malformações Arteriovenosas/metabolismo , Encéfalo/fisiopatologia , Proteínas Hedgehog/metabolismo , Animais , HumanosRESUMO
The cranial window (CW) technique provides a simple and low-cost method to assess tumor angiogenesis in the brain. The CW combined with histology using selective markers for tumor and endothelial cells can allow a sensitive monitoring of novel antiangiogenesis therapies in preclinical models. The CW was established in cyclosporine immunosuppressed rats that were stereotactically grafted with fluorescent U87MG glioblastoma cells. One to 3 weeks after grafting, brain vasculature was visualized in vivo and assessed by immunofluorescence microscopy using antibodies against endothelial and smooth-muscle cells and blood brain barrier. At 1-2 weeks after grafting, the CW reliably detected the hypertrophy of venous-venous anastomoses and cortical veins. These structures increased highly significantly their pregrafting diameter. Arterialized veins and hemorrhages were seen by three weeks after grafting. Immunofluorescence microscopy showed significant branching and dilation of microvessels, particularly those surrounded by tumor cells. Mechanistically, these changes lead to loss of vascular resistance, increased venous outflow, and opening of venous-venous anastomoses on the cortical surface. Data from the present study, namely, the hypertrophy of cortical venous-venous anastomoses, microvessel branching, and dilation of the microvessels surrounded by tumor cells, indicate the power of this in vivo model for the sensitive monitoring of early tumor angiogenesis.
Assuntos
Bioensaio , Neoplasias Encefálicas , Encéfalo , Veias Cerebrais , Glioblastoma , Neoplasias Experimentais , Neovascularização Patológica , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Veias Cerebrais/metabolismo , Veias Cerebrais/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ratos , Ratos WistarRESUMO
Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.
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Glioblastoma/genética , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , HumanosRESUMO
In the CNS, endocannabinoids are identified mainly as two endogenous lipids: anandamide, the ethanolamide of arachidonic acid, and 2-arachidonoylglycerol (2-AG). Endocannabinoids are known to inhibit transmitter release from presynaptic terminals; however we have recently demonstrated that they are also involved in slow self-inhibition (SSI) of layer V low-threshold spiking (LTS) interneurons in rat somatosensory cortex. SSI is induced by repetitive firing in LTS cells, which can express either cholecystokinin or somatostatin. SSI is triggered by an endocannabinoid-dependent activation of a prolonged somatodendritic K(+) conductance and associated hyperpolarization in the same cell. The synthesis of both endocannabinoids is dependent on elevated [Ca(2+)](i) such as occurs during sustained neuronal activity. To establish whether 2-AG mediates autocrine LTS-SSI, we blocked its biosynthesis from phospholipase C (PLC) and diacylglycerol lipases (DAGLs). Current-clamp recordings from LTS interneurons in acute neocortical slices showed that inclusion of DAGL inhibitors in the whole-cell pipette prevented the long-lasting hyperpolarization triggered by LTS cell repetitive firing. Similarly, extracellular applications of a PLC inhibitor prevented SSI in LTS interneurons. Moreover, metabotropic glutamate receptor-dependent activation of PLC produced a long-lasting hyperpolarization which was prevented by the CB1 antagonist AM251, as well as by PLC and DAGL inhibitors. The loss of SSI in the presence of intracellular DAGL blockers confirms that endocannabinoid production occurs in the same interneuron undergoing the persistent hyperpolarization. Since DAGLs produce no endocannabinoid other than 2-AG, these results identify this compound as the autocrine mediator responsible for the postsynaptic slow self-inhibition of neocortical LTS interneurons.
Assuntos
Ácidos Araquidônicos/metabolismo , Glicerídeos/metabolismo , Interneurônios/metabolismo , Neocórtex/metabolismo , Inibição Neural/fisiologia , Animais , Endocanabinoides , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Interneurônios/efeitos dos fármacos , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/efeitos dos fármacos , Neocórtex/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
Inhibitors of Vascular Endothelial Growth Factor target both tumor vasculature and cancer cells that have hijacked VEGF Receptors (VEGFRs) signaling for tumor growth-promoting activities. It is important to get precise insight in the specificity of cell responses to these antiangiogenic drugs to maximize their efficiency and minimize off-target systemic toxicity. Here we report that Axitinib, an inhibitor of VEGFRs currently in use as a second line treatment for advanced renal cell carcinoma, promotes senescence of human endothelial cells in vitro. A one-hour pulse of Axitinib is sufficient for triggering cell senescence. Mechanistically, this requires oxidative stress-dependent activation of the Ataxia Telangiectasia Mutated (ATM) kinase. Axitinib-mediated senescence promoting action is prevented by short-term treatment with antioxidants or ATM inhibitors, which conversely fail to prevent senescence induced by the DNA-damaging drug doxorubicin. Coherently, induction of oxidative stress-related genes distinguishes the response of endothelial cells to Axitinib from that to doxorubicin. Importantly, an Axitinib pulse causes cell senescence in glioblastoma cells. However, neither antioxidants nor ATM inhibitors can reverse this phenotype. Thus, antioxidants may selectively protect endothelial cells from Axitinib by decreasing systemic toxicity and maintaining a functional vascularization necessary for efficient delivery of chemotherapeutic drugs within the tumor mass.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Axitinibe/farmacologia , Senescência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Angiogênese/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Células Endoteliais/metabolismo , Ativação Enzimática , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Patológica/prevenção & controle , Inibidores de Proteínas Quinases/administração & dosagemRESUMO
The question whether perivascular glioma cells invading the brain far from the tumor bulk may disrupt the blood-brain barrier (BBB) represents a crucial issue because under this condition tumor cells would be no more protected from the reach of chemotherapeutic drugs. A recent in vivo study that used human xenolines, demonstrated that single glioma cells migrating away from the tumor bulk are sufficient to breach the BBB. Here, we used brain xenografts of patient-derived glioma stem-like cells (GSCs) to show by immunostaining that in spite of massive perivascular invasion, BBB integrity was preserved in the majority of vessels located outside the tumor bulk. Interestingly, the tumor cells that invaded the brain for the longest distances traveled along vessels with retained BBB integrity. In surgical specimens of malignant glioma, the area of brain invasion showed several vessels with preserved BBB that were surrounded by tumor cells. On transmission electron microscopy, the cell inter-junctions and basal lamina of the brain endothelium were preserved even in conditions in which the tumor cells lay adjacently to blood vessels. In conclusion, BBB integrity associates with extensive perivascular invasion of glioma cells.
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The role of autophagy in cancer onset and progression appears still controversial. On one hand, autophagy allows cancer cell to survive in unfavorable environmental conditions, on the other hand, once internal energy resources are exhausted, it leads to cell death. In addition, autophagy interpheres with cell cycle progression, de facto exerting a cytostatic activity. Hence, it represents an important target for anticancer therapy. For example, temozolomide (TMZ), of use for glioblastoma (GBM) treatment, appears as capable of inducing autophagy partially inhibiting cancer cell proliferation. However, GBM, a very aggressive brain tumor with poor prognosis even after surgery and radio-chemotherapy, invariably recurs and leads to patient death. Since cancer stem cells have been hypothesized to play a role in refractory/relapsing cancers, in the present work we investigated if autophagy could represent a constitutive cytoprotection mechanism for glioblastoma stem-like cells (GSCs) and if the modulation of autophagic process could affect GBM growth and survival. Thus, in the present study we first evaluated the relevance of autophagy in GBM tumor specimens, then its occurrence in GSCs and, finally, if modulation of autophagy could influence GSC response to TMZ. Our results suggested that, in vitro, the impairing autophagic process with quinacrine, a compound able to cross the blood-brain barrier, increased GSC susceptibility to TMZ. Death of GSCs was apparently due to the iron dependent form of programmed cell death characterized by the accumulation of lipid peroxides called ferroptosis. These results underscore the relevance of the modulation of autophagy in the GSC survival and death and suggest that triggering of ferroptosis in GSCs could represent a novel and important target for the management of glioblastoma.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Temozolomida/farmacologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/mortalidade , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Quinacrina/farmacologia , Quinacrina/uso terapêutico , Proteína Sequestossoma-1/metabolismo , Temozolomida/uso terapêutico , Transplante HeterólogoRESUMO
BACKGROUND: Mesenchymal stem/stromal cells (MSCs) represent an attractive tool for cell-based cancer therapy mainly because of their ability to migrate to tumors and to release bioactive molecules. However, the impact of MSCs on tumor growth has not been fully established. We previously demonstrated that murine MSCs show a strong tropism towards glioblastoma (GBM) brain xenografts and that these cells are able to uptake and release the chemotherapeutic drug paclitaxel (PTX), maintaining their tropism towards the tumor. Here, we address the therapy-relevant issue of using MSCs from human donors (hMSCs) for local or systemic administration in orthotopic GBM models, including xenografts of patient-derived glioma stem cells (GSCs). METHODS: U87MG or GSC1 cells expressing the green fluorescent protein (GFP) were grafted onto the striatum of immunosuppressed rats. Adipose hMSCs (Ad-hMSCs), fluorescently labeled with the mCherry protein, were inoculated adjacent to or into the tumor. In rats bearing U87MG xenografts, systemic injections of Ad-hMSCs or bone marrow (BM)-hMSCs were done via the femoral vein or carotid artery. In each experiment, either PTX-loaded or unloaded hMSCs were used. To characterize the effects of hMSCs on tumor growth, we analyzed survival, tumor volume, tumor cell proliferation, and microvascular density. RESULTS: Overall, the AD-hMSCs showed remarkable tropism towards the tumor. Intracerebral injection of Ad-hMSCs significantly improved the survival of rats with U87MG xenografts. This effect was associated with a reduction in tumor growth, tumor cell proliferation, and microvascular density. In GSC1 xenografts, intratumoral injection of Ad-hMSCs depleted the tumor cell population and induced migration of resident microglial cells. Overall, PTX loading did not significantly enhance the antitumor potential of hMSCs. Systemically injected Ad- and BM-hMSCs homed to tumor xenografts. The efficiency of hMSC homing ranged between 0.02 and 0.5% of the injected cells, depending both on the route of cell injection and on the source from which the hMSCs were derived. Importantly, systemically injected PTX-loaded hMSCs that homed to the xenograft induced cytotoxic damage to the surrounding tumor cells. CONCLUSIONS: hMSCs have a therapeutic potential in GBM brain xenografts which is also expressed against the GSC population. In this context, PTX loading of hMSCs seems to play a minor role.
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Proliferação de Células , Glioblastoma/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Linhagem Celular Tumoral , Terapia Combinada , Glioblastoma/patologia , Humanos , Camundongos , Paclitaxel/administração & dosagem , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: The goal of cancer chemotherapy is targeting tumor cells and/or tumor-associated microvessels with the lowest systemic toxicity. Mesenchymal stromal cells (MSCs) are promising vehicles for selective drug delivery due to their peculiar ability to home to pathological tissues. We previously showed that MSCs are able to uptake and subsequently to release the chemotherapeutic compound Paclitaxel (PTX) and to impair the growth of subcutaneous glioblastoma multiforme (GBM) xenografts. Here we used an orthotopic GBM model 1) to assess whether PTX-loaded MSCs (PTX-MSCs) retain a tropism towards the tumor cells in the brain context, and 2) to characterize the cytotoxic damage induced by MSCs-driven PTX release in the tumor microenvironment. METHODS: U87MG GBM cells were fluorescently labeled with the mCherry protein and grafted onto the brain of immunosuppressed rats. In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded. After 1 week survival, the xenografted brain was assessed by confocal microscopy for PTX-induced cell damage. RESULTS: Overall, MSCs showed remarkable tropism towards the tumor. In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation. Multi-spindle mitoses resulted in multinucleated cells that were significantly higher in tumors co-grafted with PTX-MSCs than in controls. Nuclear changes did not occur in astrocytes and neurons surrounding the tumor. CONCLUSIONS: MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Transplante de Células-Tronco Mesenquimais , Paclitaxel/administração & dosagem , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos Fitogênicos/toxicidade , Linhagem Celular Tumoral , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Paclitaxel/uso terapêutico , Paclitaxel/toxicidade , Ratos , Ratos WistarRESUMO
Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.
Assuntos
Neoplasias do Sistema Nervoso Central/patologia , Proteínas de Ligação a DNA/metabolismo , Glioblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição/metabolismo , Angiopoietina-2/metabolismo , Animais , Biomarcadores/metabolismo , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Janus Quinase 1/metabolismo , Janus Quinase 1/farmacologia , Camundongos , Células-Tronco Neoplásicas/patologia , Fosforilação , RNA Interferente Pequeno , Proteínas Repressoras , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The adult hippocampus is involved in learning and memory. As a consequence, it is a brain region of remarkable plasticity. This plasticity exhibits itself both as cellular changes and neurogenesis. For neurogenesis to occur, a population of local stem cells and progenitor cells is maintained in the adult brain and these are able to proliferate and differentiate into neurons which contribute to the hippocampal circuitry. There is much interest in understanding the role of immature cells in the hippocampus, in relation to learning and memory. Methods and mechanisms that increase the numbers of these cells will be valuable in this research field. We show here that single injections of soluble factors into the lateral ventricle of adult rats and mice induces the rapid (within one week) increase in the number of putative stem cells/progenitor cells in the hippocampus. The established progenitor marker Sox2 together with the more recently established marker Hes3, were used to quantify the manipulation of the Sox2/Hes3 double-positive cell population. We report that in both adult rodent species, Sox2+/Hes3+ cell numbers can be increased within one week. The most prominent increase was observed in the hilus of the dentate gyrus. This study presents a fast, pharmacological method to manipulate the numbers of endogenous putative stem cells/progenitor cells. This method may be easily modified to alter the degree of activation (e.g. by the use of osmotic pumps for delivery, or by repeat injections through implanted cannulas), in order to be best adapted to different paradigms of research (neurodegenerative disease, neuroprotection, learning, memory, plasticity, etc).
Assuntos
Envelhecimento/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hipocampo/citologia , Proteínas Recombinantes/farmacologia , Fatores de Transcrição SOXB1/metabolismo , Animais , Animais Recém-Nascidos , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Hipocampo/anatomia & histologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , SolubilidadeRESUMO
Synaptic loss is the best pathological correlate of the cognitive decline in Alzheimer's disease; however, the molecular mechanisms underlying synaptic failure are unknown. We found a non-apoptotic baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this activity at the onset of memory decline in the Tg2576-APPswe mouse model of Alzheimer's disease. In spines, caspase-3 activated calcineurin, which in turn triggered dephosphorylation and removal of the GluR1 subunit of AMPA-type receptor from postsynaptic sites. These molecular modifications led to alterations of glutamatergic synaptic transmission and plasticity and correlated with spine degeneration and a deficit in hippocampal-dependent memory. Notably, pharmacological inhibition of caspase-3 activity in Tg2576 mice rescued the observed Alzheimer-like phenotypes. Our results identify a previously unknown caspase-3-dependent mechanism that drives synaptic failure and contributes to cognitive dysfunction in Alzheimer's disease. These findings indicate that caspase-3 is a potential target for pharmacological therapy during early disease stages.
Assuntos
Doença de Alzheimer/metabolismo , Caspase 3/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Transmissão Sináptica/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Calcineurina/metabolismo , Inibidores de Caspase , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Transtornos da Memória/genética , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Transgênicos , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Oligopeptídeos/farmacologia , Ácido Poliglutâmico/farmacologia , Receptores de AMPA/metabolismoRESUMO
Control of pyramidal neuron excitability is vital for the functioning of the neocortex. Somatodendritic slow self-inhibition (SSI) allows inhibitory neurons to regulate their own activity, but the existence of similar mechanisms in excitatory cells has not been shown. We found that in rodents endocannabinoids mediated SSI and long-term modulation of inhibitory connections in layer 2/3 pyramidal neurons with a distinct dendritic morphology, suggesting that a glutamatergic network in cortical circuits is self-regulated.