RESUMO
Resistance to antiangiogenic therapy in glioblastoma (GBM) patients may involve hypoxia-induced expression of C-X-C motif chemokine receptor 4 (CXCR4) on invading tumor cells, macrophage/microglial cells (MGCs), and glioma stem cells (GSCs). We determined whether antagonizing CXCR4 with POL5551 disrupts anti-vascular endothelial growth factor (VEGF) therapy-induced glioma growth and dissemination. Mice bearing orthotopic CT-2A or GL261 gliomas received POL5551 and/or anti-VEGF antibody B20-4.1.1. Brain tissue was analyzed for tumor volume, invasiveness, hypoxia, vascular density, proliferation, apoptosis, GSCs, and MGCs. Glioma cells were evaluated for CXCR4 expression and polymorphism and POL5551's effects on CXCR4 ligand binding, cell viability, and migration. No CXCR4 mutations were identified. POL5551 inhibited CXCR4 binding to its ligand, stromal cell-derived factor-1α, and reduced hypoxia- and stromal cell-derived factor-1α-mediated migration dose-dependently but minimally affected cell viability. In vivo, B20-4.1.1 increased hypoxic foci and invasiveness, as seen in GBM patients receiving anti-VEGF therapy. Combination of POL5551 and B20-4.1.1 reduced both glioma invasiveness by 16% to 39% and vascular density compared to B20-4.1.1 alone in both glioma models. Reduced populations of GSCs and MGCs were also seen in CT-2A tumors. POL5551 concentrations, evaluated by mass spectrometry, were higher in tumors than in neighboring brain tissues, likely accounting for the results. Inhibition of CXCR4-regulated tumoral, stem cell, and immune mechanisms by adjunctive CXCR4 antagonists may help overcome antiangiogenic therapy resistance, benefiting GBM patients.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos/uso terapêutico , Glioma/tratamento farmacológico , Receptores CXCR4/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glioma/metabolismo , Glioma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion. METHODS: In order to perform the studies, we employed optimal cell culture methods and hypoxic conditions, lentivirus-mediated knockdown of protein expression, Western Blot analysis, migration assays and immunoprecipitation. We determined statistical significance by unpaired t-test. RESULTS: In this report, we show that U87MG, LN229 and LN308 glioma cells express CXCR7 and that exposure to hypoxia upregulates CXCR7 protein expression in these cell lines. CXCR7-expressing U87MG, LN229 and LN308 glioma cells migrated towards stromal-derived factor (SDF)-1α/CXCL12 in hypoxic conditions in the Boyden chamber assays. While shRNA-mediated knockdown of CXCR7 expression did not affect the migration of any of the three cell lines in normoxic conditions, we observed a reduction in the migration of LN229 and LN308, but not U87MG, glioma cells towards SDF-1α in hypoxic conditions. In addition, knockdown of CXCR7 expression in LN229 and LN308 glioma cells decreased levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. Inhibiting CXCR4 in LN229 and LN308 glioma cells that were knocked down for CXCR7 did not further reduce migration towards SDF-1α in hypoxic conditions and did not affect the levels of phosphorylated ERK1/2 and Akt. Analysis of immunoprecipitated CXCR4 from LN229 and LN308 glioma cells revealed co-precipitated CXCR7. CONCLUSIONS: Taken together, our findings indicate that both CXCR4 and CXCR7 mediate glioma cell migration towards SDF-1α in hypoxic conditions and support the development of therapeutic agents targeting these receptors.
Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Glioma/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Western Blotting , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Técnicas de Silenciamento de Genes , Glioma/patologia , Humanos , Imunoprecipitação , Invasividade Neoplásica/patologiaRESUMO
The present study examined the ability of 6-fluoronorepinephrine (6FNE), a full selective α-adrenoceptor agonist, to produce antidepressant-like effects in mice. The drug, administered in the 4th ventricle, produced marked anti-immobility effects at mid-dose range in the acute forced swim, tail suspension and repeated open-space forced swim tests with minimal effect on open-field motor activity and also reversed anhedonia following lipopolysaccharide administration. Its antidepressant effects were equal to or greater than that of an established systemic antidepressant, desmethylimipramine, given subacutely. Experiments with α-adrenoceptor antagonists indicated that the drug acts primarily via the α2-receptor in contrast to endogenous catecholamines which appear to control depressive behaviour primarily via the α1-receptor. Antidepressant activity declined at higher doses signifying a possible pro-depressant effect of one of the α-adrenoceptor subtypes. Compared to the selective α2-agonist, dexmedetomidine, 6FNE showed equivalent antidepressant action in the tail suspension test but appeared to have a greater efficacy or speed of action in the repeated open-space forced swim test which produces a more sustained depression. Studies of regional brain Fos expression induced during the antidepressant tests showed that 6FNE tended to inhibit neural activity in two stress-responsive regions (locus coeruleus and paraventricular hypothalamus) but to enhance activity in two areas involved in motivated behaviour (nucleus accumbens shell and lateral septal nucleus) producing a neural pattern consistent with antidepressant action. It is concluded that 6FNE elicits a rapid and effective antidepressant and anti-stress response that may compare favourably with available antidepressants.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antidepressivos/farmacologia , Depressão/tratamento farmacológico , Desipramina/farmacologia , Norepinefrina/análogos & derivados , Agonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Animais , Antidepressivos/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Elevação dos Membros Posteriores , Injeções Intraventriculares , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Norepinefrina/administração & dosagem , Norepinefrina/farmacologia , Prazer/efeitos dos fármacos , Distribuição Aleatória , NataçãoRESUMO
This study was designed to replicate an earlier finding of a rapid acute therapeutic action of intracerebrally administered antidepressant in chronically depressed rodents. The effects of acute fourth ventricular (ivt.) injections were compared to those of acute peripheral (i.p.) injections of desipramine (DMI) in mice subjected to repeated open-space forced swim. In confirmation, it was found that a single ivt. injection of a low (3 nmol) but not high (30 nmol) dose immediately reversed the immobility and inactivity of the model whereas acute i.p. administration was without effect up to 30 mg/kg. The repeated forced swim stress was also found to significantly reduce the net accumulation of DMI in the brain but not liver after a single i.p. injection of a moderate dose (10 mg/kg). The results suggest that stress-induced alterations of regional drug uptake or metabolism in the CNS may contribute to the therapeutic lag for antidepressants and other compounds in disorders with high distress.
Assuntos
Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Depressão/tratamento farmacológico , Transtorno Depressivo/tratamento farmacológico , Animais , Antidepressivos/administração & dosagem , Antidepressivos/uso terapêutico , Encéfalo/efeitos dos fármacos , Desipramina/administração & dosagem , Desipramina/farmacologia , Desipramina/uso terapêutico , Injeções , Injeções Intraventriculares , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Natação , Fatores de TempoRESUMO
Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. In vivo, RT-TEX elicited tumor-specific CD8+ T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. Cancer Immunol Res; 6(8); 910-20. ©2018 AACR.
Assuntos
DNA de Neoplasias/imunologia , Células Dendríticas/imunologia , Exodesoxirribonucleases/imunologia , Exossomos/genética , Neoplasias Mamárias Animais/imunologia , Fosfoproteínas/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Exossomos/imunologia , Feminino , Interferon Tipo I/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/prevenção & controle , Neoplasias Mamárias Animais/radioterapia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/imunologia , Células Tumorais CultivadasRESUMO
Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12-18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-ß by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-ß production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/efeitos da radiação , Neoplasias Mamárias Animais/genética , Neoplasias/imunologia , Neoplasias/radioterapia , Fosfoproteínas/metabolismo , Fosfoproteínas/efeitos da radiação , Animais , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Linfócitos T CD8-Positivos/efeitos da radiação , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos da radiação , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Imunoterapia/métodos , Interferon beta/metabolismo , Interferon beta/efeitos da radiação , Neoplasias Mamárias Animais/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Nucleotidiltransferases/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Proteínas Repressoras/imunologiaRESUMO
INTRODUCTION: Inflammatory breast cancer (IBC) is an aggressive and rare cancer with a poor prognosis and a need for novel targeted therapeutic strategies. Preclinical IBC data showed strong activation of the phosphatidylinositide-3-kinase/mammalian target of rapamycin (mTOR) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways, and expression of inflammatory cytokines and tumor-associated macrophages (TAMs). PATIENTS AND METHODS: Archival tumor tissue from 3 disease types (IBC treated with neoadjuvant chemotherapy [NAC], n = 45; invasive ductal carcinoma [IDC] treated with NAC [n = 24; 'treated IDC'; and untreated IDC [n = 27; 'untreated IDC']) was analyzed for the expression of biomarkers phospho-S6 (pS6) (mTOR), phospho-JAK2 (pJAK2), pSTAT3, interleukin (IL)-6, CD68 (monocytes, macrophages), and CD163 (TAMs). Surrounding nontumor tissue was also analyzed. RESULTS: Biomarker levels and surrogate activity according to site-specific phosphorylation were shown in the tumor tissue of all 3 disease types but were greatest in IBC and treated IDC and least in untreated IDC for pS6, pJAK2, pSTAT3, and IL-6. Of 37 IBC patients with complete biomarker data available, 100% were pS6-positive and 95% were pJAK2-positive. In nontumor tissue, biomarker levels were observed in all groups but were generally greatest in untreated IDC and least in IBC, except for JAK2. CONCLUSION: IBC and treated IDC display similar levels of mTOR and JAK2 biomarker activation, which suggests a potential mechanism of resistance after NAC. Biomarker levels in surrounding nontumor tissue suggested that the stroma might be activated by chemotherapy and resembles the oncogenic tumor-promoting environment. Activation of pS6 and pJAK2 in IBC might support dual targeting of the mTOR and JAK/STAT pathways, and the need for prospective studies to investigate combined targeted therapies in IBC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Neoplasias Inflamatórias Mamárias/patologia , Janus Quinase 2/metabolismo , Terapia Neoadjuvante , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/metabolismo , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Neoplasias Inflamatórias Mamárias/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxa de SobrevidaRESUMO
UNLABELLED: Cystathionine ß-synthase (CBS) catalyzes metabolic reactions that convert homocysteine to cystathionine. To assess the role of CBS in human glioma, cells were stably transfected with lentiviral vectors encoding shRNA targeting CBS or a nontargeting control shRNA, and subclones were injected into immunodeficient mice. Interestingly, decreased CBS expression did not affect proliferation in vitro but decreased the latency period before rapid tumor xenograft growth after subcutaneous injection and increased tumor incidence and volume following orthotopic implantation into the caudate-putamen. In soft-agar colony formation assays, CBS knockdown subclones displayed increased anchorage-independent growth. Molecular analysis revealed that CBS knockdown subclones expressed higher basal levels of the transcriptional activator hypoxia-inducible factor 2α (HIF2α/EPAS1). HIF2α knockdown counteracted the effect of CBS knockdown on anchorage-independent growth. Bioinformatic analysis of mRNA expression data from human glioma specimens revealed a significant association between low expression of CBS mRNA and high expression of angiopoietin-like 4 (ANGPTL4) and VEGF transcripts, which are HIF2 target gene products that were also increased in CBS knockdown subclones. These results suggest that decreased CBS expression in glioma increases HIF2α protein levels and HIF2 target gene expression, which promotes glioma tumor formation. IMPLICATIONS: CBS loss-of-function promotes glioma growth.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Carcinogênese/patologia , Cistationina beta-Sintase/metabolismo , Glioma/enzimologia , Glioma/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Cistationina beta-Sintase/deficiência , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Injeções Subcutâneas , Masculino , Camundongos SCID , Carga Tumoral , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although the central noradrenergic system has been shown to be involved in a number of behavioral and neurophysiological processes, the relation of these to its role in depressive illness has been difficult to define. The present review discusses the hypothesis that one of its chief functions that may be related to affective illness is the inhibition of behavioral activation, a prominent symptom of the disorder. This hypothesis is found to be consistent with most previous neuropsychopharmacological and immunohistochemical experiments on active behavior in rodents in a variety of experimental conditions using manipulation of neurotransmission at both locus coeruleus and forebrain adrenergic receptors. The findings support a mechanism in which high rates of noradrenergic neural activity suppress the neural activity of principal neurons in forebrain regions mediating active behavior. The suppression may be mediated through postsynaptic galaninergic and adrenergic receptors, and via the release of corticotrophin-releasing hormone. The hypothesis is consistent with clinical evidence for central noradrenergic system hyperactivity in depressives and with the view that this hyperactivity is a contributing etiological factor in the disorder. A similar mechanism may underlie the ability of the noradrenergic system to suppress seizure activity suggesting that inhibition of the spread of neural activation may be a unifying function.
Assuntos
Neurônios Adrenérgicos/fisiologia , Transtorno Depressivo/patologia , Inibição Psicológica , Locus Cerúleo/patologia , Neurônios Adrenérgicos/patologia , Animais , Transtorno Depressivo/psicologia , Humanos , Locus Cerúleo/citologia , Locus Cerúleo/metabolismo , Inibição Neural/fisiologia , Vias Neurais/citologia , Vias Neurais/patologiaRESUMO
Several lines of research have now suggested the controversial hypothesis that the central noradrenergic system acts to exacerbate depression as opposed to having an antidepressant function. If correct, lesions of this system should increase resistance to depression, which has been partially but weakly supported by previous studies. The present study reexamined this question using two more recent methods to lesion noradrenergic neurons in mice: intraventricular (ivt) administration of either the noradrenergic neurotoxin, DSP4, or of a dopamine-ß-hydroxylase-saporin immunotoxin (DBH-SAP ITX) prepared for mice. Both agents given 2 weeks prior were found to significantly increase resistance to depressive behavior in several tests including acute and repeated forced swims, tail suspension and endotoxin-induced anhedonia. Both agents also increased locomotor activity in the open field. Cell counts of brainstem monoaminergic neurons, however, showed that both methods produced only partial lesions of the locus coeruleus and also affected the dorsal raphe or ventral tegmental area. Both the cell damage and the antidepressant and hyperactive effects of ivt DSP4 were prevented by a prior i.p. injection of the NE uptake blocker, reboxetine. The results are seen to be consistent with recent pharmacological experiments showing that noradrenergic and serotonergic systems function to inhibit active behavior. Comparison with previous studies utilizing more complete and selective LC lesions suggest that mouse strain, lesion size or involvement of multiple neuronal systems are critical variables in the behavioral and affective effects of monoaminergic lesions and that antidepressant effects and hyperactivity may be more likely to occur if lesions are partial and/or involve multiple monoaminergic systems.
Assuntos
Monoaminas Biogênicas/fisiologia , Tronco Encefálico/metabolismo , Núcleo Celular/metabolismo , Depressão/metabolismo , Animais , Monoaminas Biogênicas/metabolismo , Cromatografia Líquida de Alta Pressão , Imuno-Histoquímica , Locomoção , Masculino , CamundongosRESUMO
alpha(1)-Adrenoceptors are concentrated in the locus coeruleus (LC) where they appear to regulate various active behaviors but have been difficult to stimulate effectively. The present study examined the behavioral, pharmacological and neural effects of possible stimulation of these receptors with 6-fluoronorepinephrine (6FNE), the only known selective alpha-agonist that has full efficacy at all brain alpha-receptors. Infusion of this compound in the mouse LC was found to produce extreme activation of diverse motivated behaviors of exploration, wheel-running and operant approach responding in different environments consistent with a global behavioral function of the dorsal noradrenergic system. Infusion of selective antagonists of alpha(1)- (terazosin) or alpha(2)- (atipamezole) receptors or of either the partial alpha(1)-agonist, phenylephrine, or full alpha(2)-agonist, dexmedetomidine, indicated that the behavioral effects of 6FNE were due largely due to activation of LC alpha(1)-receptors consistent with the known greater density of alpha(1)- than alpha(2)-adrenoreceptors in the mouse nucleus. Immunohistochemistry of fos in tyrosine hydroxylase-positive LC neurons following IV ventricular infusions indicated that 6FNE markedly depressed whereas terazosin strongly enhanced the apparent functional activity of the nucleus. The changes in fos expression following 6FNE and terazosin were significantly greater than those following dexmedetomidine and atipamezole. It is hypothesized that the alpha(1)-receptors of the mouse LC are strongly activated by 6FNE and serve to potently inhibit its tonic or stress-induced activity which in turn disinhibits prepotent motivated behaviors.