RESUMO
Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.
Assuntos
Complemento C5a/imunologia , Armadilhas Extracelulares/imunologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Imunofenotipagem , Camundongos , Modelos Biológicos , Metástase Neoplásica , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/patologia , Receptor da Anafilatoxina C5a/metabolismoRESUMO
Microvascular injury during acute rejection has been associated with massive infiltration of CD4+ T effector cells, and the formation of complement products (C3a and C5a). Regulatory T cells (Tregs) are potent immunosuppressors of the adaptive immune system and have proven sufficient to rescue microvascular impairments. Targeting C5a has been linked with improved microvascular recovery, but its effects on the Treg and T effector balance is less well known. Here, we demonstrate the impact of C5a blockade on Treg induction and microvascular restoration in rejecting mouse airway allografts. BALB/câC57BL/6 allografts were treated with a C5a-neutralizing l-aptamer (10 mg/kg, i.p. at d0 and every second day thereafter), and allografts were serially monitored for Treg infiltration, tissue oxygenation (tpO2), microvascular blood flow, and functional microvasculature between donor and recipients during allograft rejection. We demonstrated that C5a blocking significantly leads to enhanced presence of Tregs in the allograft, reinstates donor-recipient functional microvasculature, improves tpO2, microvascular blood flow, and epithelial repair, followed by an upregulation of IL-5, TGF-ß, IL-10 vascular endothelial growth factor, and ANGPT1 gene expression, while it maintained a healthy epithelium and prevented subepithelial collagen deposition at d28 posttransplantation. Together, these data indicate that inhibition of C5a signaling has potential to preserve microvasculature and rescue allograft from a sustained hypoxic/ischemic phase, limits airway tissue remodeling through the induction of Treg-mediated immune tolerance. These findings may be useful in designing anti-C5a therapy in combination with existing immunosuppressive regimens to rescue tissue/organ rejection.
Assuntos
Complemento C5a/antagonistas & inibidores , Células Epiteliais/imunologia , Células Epiteliais/patologia , Rejeição de Enxerto/prevenção & controle , Linfócitos T Reguladores/imunologia , Traqueia/transplante , Aloenxertos/imunologia , Animais , Complemento C5a/imunologia , Rejeição de Enxerto/imunologia , Tolerância Imunológica , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microvasos/imunologia , Sistema Respiratório/citologia , Sistema Respiratório/imunologia , Traqueia/imunologia , Transplante HomólogoRESUMO
Immune checkpoint inhibitors promote T cell-mediated killing of cancer cells; however, only a subset of patients benefit from the treatment. A possible reason for this limitation may be that the tumor microenvironment (TME) is immune privileged, which may exclude cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 is key to the TME-driven immune suppression. In this study, we investigated the potential of CXCL12 inhibition by use of the clinical-stage l-RNA-aptamer NOX-A12 (olaptesed pegol) to increase the number of tumor-infiltrating lymphocytes. We used heterotypic tumor-stroma spheroids that mimic a solid tumor with a CXCL12-abundant TME. NOX-A12 enhanced the infiltration of T and NK cells in a dose-dependent manner. NOX-A12 and PD-1 checkpoint inhibition synergistically activated T cells in the spheroids, indicating that the agents complement each other. The findings were validated in vivo in a syngeneic murine model of colorectal cancer in which the addition of NOX-A12 improved anti-PD-1 therapy. Taken together, our work shows that CXCL12 inhibition can break the immune-privileged status of the TME by paving the way for immune effector cells to enter into the tumor, thereby broadening the applicability of checkpoint inhibitors in cancer patients. Cancer Immunol Res; 5(11); 950-6. ©2017 AACR.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Quimiocina CXCL12/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos Imunológicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Quimiocina CXCL12/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Microambiente Tumoral/imunologiaRESUMO
Several mechanisms have been postulated for orchestrating the mobilization of hematopoietic stem/progenitor cells (HSPCs), and we previously proposed that activation of the complement cascade plays a crucial role in the initiation and execution of the egress of HSPCs from bone marrow (BM) into peripheral blood (PB). In support of this notion, we demonstrated that mice deficient in the mannan-binding lectin (MBL) pathway, which activates the proximal part of the complement cascade, as well as mice deficient in the fifth component of the complement cascade (C5), which is part of the distal part of the complement cascade, are poor mobilizers. To further narrow down on the exact mechanisms and the molecules involved, we performed studies in mice that do not express the receptor C5aR, which binds the C5 cleavage fragments, C5a and C5adesArg. We also employed the plasma stable nucleic acid aptamer AON-D21 that binds and neutralizes C5a and C5adesArg. We present evidence that mice deficient in C5aR or treated with AON-D21 are poor HSPC mobilizers, thereby establishing a critical role for the C5a/C5adesArg-C5aR axis in the mobilization process. While enhancing mobilization is of clinical importance for poor mobilizers, inhibition of the complement cascade could be of therapeutic importance in patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) or acquired hemolytic syndrome (aHUS).
Assuntos
Complemento C5a/genética , Células-Tronco Hematopoéticas/citologia , Lectina de Ligação a Manose/genética , Receptor da Anafilatoxina C5a/genética , Anafilatoxinas/genética , Animais , Ativação do Complemento/genética , Complemento C5a des-Arginina/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinúria Paroxística , Humanos , Lectina de Ligação a Manose/deficiência , CamundongosRESUMO
Disruption of the programmed cell death protein 1 (PD-1) pathway with immune checkpoint inhibitors represents a major breakthrough in the treatment of non-small cell lung cancer. We hypothesized that combined inhibition of C5a/C5aR1 and PD-1 signaling may have a synergistic antitumor effect. The RMP1-14 antibody was used to block PD-1, and an L-aptamer was used to inhibit signaling of complement C5a with its receptors. Using syngeneic models of lung cancer, we demonstrate that the combination of C5a and PD-1 blockade markedly reduces tumor growth and metastasis and leads to prolonged survival. This effect is accompanied by a negative association between the frequency of CD8 T cells and myeloid-derived suppressor cells within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. Our study provides support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer.Significance: Using a variety of preclinical models of lung cancer, we demonstrate that the blockade of C5a results in a substantial improvement in the efficacy of anti-PD-1 antibodies against lung cancer growth and metastasis. This study provides the preclinical rationale for the combined blockade of PD-1/PD-L1 and C5a to restore antitumor immune responses, inhibit tumor cell growth, and improve outcomes of patients with lung cancer. Cancer Discov; 7(7); 694-703. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Complemento C5a/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Aptâmeros de Peptídeos/imunologia , Aptâmeros de Peptídeos/uso terapêutico , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Complemento C5a/imunologia , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Metástase Neoplásica , Receptor de Morte Celular Programada 1/imunologia , Transdução de SinaisRESUMO
Inflammatory events occurring in dystrophic muscles contribute to the progression of Duchenne muscular dystrophy (DMD). Low-intensity training (LIT) attenuates the phenotype of mdx mice, an animal model for DMD. Therefore, we postulated that LIT could have anti-inflammatory properties. We assessed levels of inflammatory cytokines and infiltrated immune cells in gastrocnemius muscle of mdx mice after LIT. We detected high levels of complement component C5a, chemokine ligand (CCL) 2, CD68+ monocytes/macrophages, and proinflammatory M1 macrophages in muscles of mdx mice. LIT decreased CCL2 levels, increased CD68+ cell numbers, and shifted the macrophage population to the regenerative M2 type. We investigated whether inhibition of C5a or CCL2 with L-aptamers could mimic the effects of LIT. Although no effect of CCL2 inhibition was detected, treatment with the C5a inhibitor, NOX-D21, rescued the phenotype of nonexercised mdx mice, but not of exercised ones. In both cases, the level of CD68+ cells increased and macrophage populations leaned toward the inflammatory M1 type. In muscles of nonexercised treated mice, the level of IL-1 receptor antagonist increased, damage decreased, and fibers were switched toward the glycolytic fast type; in muscles of exercised mice, fibers were switched to the oxidative slow type. These results reveal the effects of LIT on the inflammatory status of mdx mice and suggest that NOX-D21 could be an anti-inflammatory drug for DMD.
Assuntos
Complemento C5a/antagonistas & inibidores , Distrofia Muscular Animal/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Quimiocina CCL2/antagonistas & inibidores , Citocinas/metabolismo , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Membro Anterior , Macrófagos/fisiologia , Masculino , Camundongos Endogâmicos mdx , Força Muscular/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/fisiopatologia , Miosite/fisiopatologia , Miosite/prevenção & controle , Fenótipo , Natação/fisiologiaRESUMO
Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP+ cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1+) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1+) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Animais , Aptâmeros de Nucleotídeos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Quimiocina CXCL12/administração & dosagem , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Injeções Intraoculares , Iodatos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Retina/citologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/fisiopatologia , Tubulina (Proteína)/metabolismo , Acuidade Visual/efeitos dos fármacosRESUMO
Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.
Assuntos
Medula Óssea/patologia , Quimiocina CXCL12/antagonistas & inibidores , Mieloma Múltiplo/terapia , Oligonucleotídeos/farmacologia , Animais , Medula Óssea/metabolismo , Neoplasias da Medula Óssea/metabolismo , Neoplasias da Medula Óssea/secundário , Ácidos Borônicos/farmacologia , Bortezomib , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Metástase Neoplásica , Oligonucleotídeos/química , Oligonucleotídeos/genética , Polietilenoglicóis/química , Pirazinas/farmacologiaRESUMO
The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated ß-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.
Assuntos
Inibidores da Angiogênese/isolamento & purificação , Aptâmeros de Nucleotídeos/farmacologia , Lisofosfolipídeos/antagonistas & inibidores , Esfingosina/análogos & derivados , Inibidores da Angiogênese/farmacologia , Animais , Arrestinas/metabolismo , Células CHO , Cálcio/metabolismo , Cricetulus , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Neovascularização Patológica , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , beta-ArrestinasRESUMO
Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2'-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Glucagon/antagonistas & inibidores , RNA/metabolismo , Animais , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/farmacocinética , Aptâmeros de Nucleotídeos/uso terapêutico , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Jejum/sangue , Glucagon/metabolismo , Teste de Tolerância a Glucose , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Evidence suggests that bioactive lipids may regulate pathophysiologic functions such as cancer cell metastasis. Therefore, we determined that the bioactive lipid chemoattractants sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) strongly enhanced the in vitro motility and adhesion of human rhabdomyosarcoma (RMS) cells. Importantly, this effect was observed at physiologic concentrations for both bioactive lipids, which are present in biologic fluids, and were much stronger than the effects observed in response to known RMS prometastatic factors such as stromal derived factors-1 (SDF-1/CXCL12) or hepatocyte growth factor/scatter factor (HGF/SF). We also present novel evidence that the levels of S1P and C1P were increased in several organs after γ-irradiation or chemotherapy, which indicates an unwanted prometastatic environment related to treatment. Critically, we found that the metastasis of RMS cells in response to S1P can be effectively inhibited in vivo with the S1P-specific binder NOX-S93 that is based on a high-affinity Spiegelmer. These data indicate that bioactive lipids play a vital role in dissemination of RMS and contribute to the unwanted side effects of radio/chemotherapy by creating a prometastatic microenvironment.
Assuntos
Antineoplásicos/uso terapêutico , Ceramidas/metabolismo , Lisofosfolipídeos/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/radioterapia , Esfingosina/análogos & derivados , Actinas/metabolismo , Animais , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Adesão Celular/efeitos dos fármacos , Adesão Celular/efeitos da radiação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Microambiente Celular/efeitos dos fármacos , Microambiente Celular/efeitos da radiação , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Rabdomiossarcoma/enzimologia , Rabdomiossarcoma/patologia , Esfingosina/metabolismoRESUMO
Monocyte/ chemoattractant protein-1/chemokine ligand (CCL) 2 and stromal cell-derived factor-1/CXCL12 both contribute to glomerulosclerosis in mice with type 2 diabetes mellitus, through different mechanisms. CCL2 mediates macrophage-related inflammation, whereas CXCL12 contributes to podocyte loss. Therefore, we hypothesized that dual antagonism of these chemokines might have additive protective effects on the progression of diabetic nephropathy. We used chemokine antagonists based on structured l-enantiomeric RNA (so-called Spiegelmers) ie, the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12. Male db/db mice, uninephrectomized at the age of 6 weeks, received injections of Spiegelmer, both Spiegelmers, nonfunctional control Spiegelmer, or vehicle from the age of 4 months for 8 weeks. Dual blockade was significantly more effective than monotherapy in preventing glomerulosclerosis. CCL2 blockade reduced glomerular leukocyte counts and renal-inducible nitric oxide synthase or IL-6 mRNA expression. CXCL12 blockade maintained podocyte numbers and renal nephrin and podocin mRNA expression. Consistently, CXCL12 blockade suppressed nephrin mRNA up-regulation in primary cultures of human glomerular progenitors induced to differentiate toward the podocyte lineage. All previously mentioned parameters were significantly improved in the dual-blockade group, which also suppressed proteinuria and was associated with the highest levels of glomerular filtration rate. Blood glucose levels and body weight were identical in all treatment groups. Dual chemokine blockade can have additive effects on the progression of diabetic kidney disease when the respective chemokine targets mediate different pathomechanisms of disease (ie, inflammation and progenitor differentiation toward the podocyte lineage).
Assuntos
Quimiocina CCL2/antagonistas & inibidores , Quimiocina CXCL12/antagonistas & inibidores , Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/prevenção & controle , Glomerulonefrite/prevenção & controle , Animais , Western Blotting , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL12/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Taxa de Filtração Glomerular , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Técnicas Imunoenzimáticas , Interleucina-6/genética , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Podócitos/metabolismo , Podócitos/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
High mobility group A1 (HMGA1) proteins belong to a group of architectural transcription factors that are overexpressed in a range of human malignancies, including pancreatic adenocarcinoma. They promote anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. Employing in vitro selection techniques against a chosen fragment of HMGA1, we have generated biostable l-RNA oligonucleotides, so-called Spiegelmers, that specifically bind HMGA1b with low nanomolar affinity. We demonstrate that the best binding Spiegelmers, NOX-A50 and NOX-f33, compete HMGA1b from binding to its natural binding partner, AT-rich double-stranded DNA. We describe a formulation method based on polyplex formation with branched polyethylenimine for efficient delivery of polyethylene glycol-modified Spiegelmers and show improved tissue distribution and persistence in mice. In a xenograft mouse study using the pancreatic cancer cell line PSN-1, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes showed an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume. Our results demonstrate that intracellular targets can be successfully addressed with a Spiegelmer using polyethylenimine-based delivery and underline the importance of HMGA1 as a therapeutic target in pancreatic cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Aptâmeros de Nucleotídeos/farmacologia , Sistemas de Liberação de Medicamentos , Proteínas HMGA/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , DNA de Neoplasias/metabolismo , Relação Dose-Resposta a Droga , Proteínas HMGA/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Ligação ProteicaRESUMO
We developed an integrated method to identify aptamers with only 10 fixed nucleotides through ligation and removal of primer binding sites within the systematic evolution of ligands by exponential enrichment (SELEX) process. This Tailored-SELEX approach was validated by identifying a Spiegelmer ('mirror-image aptamer') that inhibits the action of the migraine-associated target calcitonin gene-related peptide 1 (alpha-CGRP) with an IC50 of 3 nM at 37 degrees C in cell culture. Aptamers are oligonucleotide ligands that can be generated to bind to targets with high affinity and specificity. Stabilized aptamers and Spiegelmers have shown activity in vivo and may be used as therapeutics. Aptamers are isolated by in vitro selection from combinatorial nucleic acid libraries that are composed of a central randomized region and additional fixed primer binding sites with approximately 30-40 nt. The identified sequences are usually not short enough for efficient chemical Spiegelmer synthesis, post-SELEX stabilization of aptamers and economical production. If the terminal primer binding sites are part of the target recognizing domain, truncation of aptamers has proven difficult and laborious. Tailored-SELEX results in short sequences that can be tested more rapidly in biological systems. Currently, our identified CGRP binding Spiegelmer serves as a lead compound for in vivo studies.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Evolução Molecular Direcionada/métodos , Transtornos de Enxaqueca/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Álcalis , Animais , Sequência de Bases , Sítios de Ligação , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Ligantes , Oligonucleotídeos/farmacologia , Reação em Cadeia da Polimerase , Ligação Proteica , Ratos , Reprodutibilidade dos Testes , Especificidade por Substrato , Transcrição GênicaRESUMO
Single-stranded mirror-image oligonucleotides, which are highly resistant to nuclease degradation and are capable of tightly and specifically binding to protein targets to inhibit their function, have been developed as potential therapeutic agents. The scientific discoveries that led to the development of the Spiegelmer technology are described in this review, along with recent preclinical developments of the first therapeutic Spiegelmers.