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The rising incidence and persistent thrombosis in multiple cancers including those that are immunosuppressive highlight the need for understanding the tumor coagulome system and its role beyond hemostatic complications. Immunotherapy has shown significant benefits in solid organ tumors but has been disappointing in the treatment of hypercoagulable cancers, such as glioblastoma and pancreatic ductal adenocarcinomas. Thus, targeting thrombosis to prevent immunosuppression seems a clinically viable approach in cancer treatment. Hypercoagulable tumors often develop fibrin clots within the tumor microenvironment (TME) that dictates the biophysical characteristics of the tumor tissue. The application of systems biology and single-cell approaches highlight the potential role of coagulome or thrombocytosis in shaping the tumor immune microenvironment (TIME). In-depth knowledge of the tumor coagulome would provide unprecedented opportunities to better predict the hemostatic complications, explore how thrombotic stroma modulates tumor immunity, reexamine the significance of clinical biomarkers, and enable steering the stromal versus systemic immune response for boosting the effectiveness of immune checkpoint inhibitors in cancer treatment. We focus on the role of coagulation factors in priming a suppressive TIME and the huge potential of existing anticoagulant drugs in the clinical settings of cancer immunotherapy.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Microambiente Tumoral , Imunoterapia/efeitos adversos , Neoplasias Pancreáticas/patologia , Terapia de Imunossupressão/efeitos adversos , Carcinoma Ductal Pancreático/patologiaRESUMO
Annually, more than a million individuals are diagnosed with gastrointestinal (GI) cancers worldwide. With the advancements in radio- and chemotherapy and surgery, the survival rates for GI cancer patients have improved in recent years. However, the prognosis for advanced-stage GI cancers remains poor. Site-specific GI cancers share a few common risk factors; however, they are largely distinct in their etiologies and descriptive epidemiologic profiles. A large number of mutations or copy number changes associated with carcinogenesis are commonly found in noncoding DNA regions, which transcribe several noncoding RNAs (ncRNAs) that are implicated to regulate cancer initiation, metastasis, and drug resistance. In this review, we summarize the regulatory functions of ncRNAs in GI cancer development, progression, chemoresistance, and health disparities. We also highlight the potential roles of ncRNAs as therapeutic targets and biomarkers, mainly focusing on their ethnicity-/race-specific prognostic value, and discuss the prospects of genome-wide association studies (GWAS) to investigate the contribution of ncRNAs in GI tumorigenesis.
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Neoplasias Gastrointestinais , MicroRNAs , RNA Longo não Codificante , Neoplasias Gastrointestinais/genética , Estudo de Associação Genômica Ampla , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA não Traduzido/genéticaRESUMO
The microfluidic-based cancer-on-a-chip models work as a powerful tool to study the tumor microenvironment and its role in metastasis. The models recapitulate and systematically simplify the in vitro tumor microenvironment. This enables the study of a metastatic process in unprecedented detail. This review examines the development of cancer-on-a-chip microfluidic platforms at the invasion/intravasation, extravasation, and angiogenesis steps over the last three years. The on-chip modeling of mechanical cues involved in the metastasis cascade are also discussed. Finally, the popular design of microfluidic chip models for each step are discussed along with the challenges and perspectives of cancer-on-a-chip models.
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Growth factors (GF) regulate normal development to cancer progression. GFs interact with extracellular matrix (ECM) biomolecules, such as heparin sulfate (HS) glycosaminoglycan (GAG), to enhance their stability and angiogenic signaling. Biomaterials that modulate GF activity by mimicking interactions observed in the native ECM could be designed as an effective treatment strategy. However, these materials failed to attenuate angiogenic signaling site-specifically without sparing normal tissues. In this work, we investigated the effect of a GAG-based biomaterial, which binds to the tumor endothelial cells (TEC), on the interaction among vascular endothelial growth factor (VEGF), its receptors-VEGFR2 and HS-and angiogenesis. Heparin-bile acid based conjugates, as ECM-mimicking component, were synthesized to selectively target the TEC marker doppel and doppel/VEGFR2 axis. The most effective compound LHbisD4 (low molecular weight heparin conjugated with 4 molecules of dimeric dexocholic acid) reduced tumor volume concentrated over doppel-expressing EC, and decreased tumor-interstitial VEGF without affecting its plasma concentration. Doppel-destined LHbisD4 captured VEGF, formed an intermediate complex with doppel, VEGFR2, and VEGF but did not induce active VEGFR2 dimerization, and competitively inhibited HS for VEGF binding. We thus show that GAG-based materials can be designed to imitate and leverage to control tumor microenvironment via bio-inspired interactions.
Assuntos
Células Endoteliais , Glicosaminoglicanos , Neoplasias , Células Endoteliais/metabolismo , Glicosaminoglicanos/farmacologia , Humanos , Neoplasias/patologia , Neovascularização Patológica/patologia , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pancreatic Ductal Adenocarcinoma (PDAC) is an expeditiously fatal malignancy with a five-year survival rate of 6-8%. Conventional chemotherapeutics fail in many cases due to inadequate primary response and rapidly developing resistance. This treatment failure is particularly challenging in pancreatic cancer because of the high molecular heterogeneity across tumors. Additionally, a rich fibro-inflammatory component within the tumor microenvironment (TME) limits the delivery and effectiveness of anticancer drugs, further contributing to the lack of response or developing resistance to conventional approaches in this cancer. As a result, there is an urgent need to model pancreatic cancer ex vivo to discover effective drug regimens, including those targeting the components of the TME on an individualized basis. Patient-derived three-dimensional (3D) organoid technology has provided a unique opportunity to study patient-specific cancerous epithelium. Patient-derived organoids cultured with the TME components can more accurately reflect the in vivo tumor environment. Here we present the advances in organoid technology and multicellular platforms that could allow for the development of "organ-on-a-chip" approaches to recapitulate the complex cellular interactions in PDAC tumors. We highlight the current advances of the organ-on-a-chip-based cancer models and discuss their potential for the preclinical selection of individualized treatment in PDAC.
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PURPOSE: Here, this study verifies that cancer-associated thrombosis (CAT) accelerates hypoxia, which is detrimental to the tumor immune microenvironment by limiting tumor perfusion. Therefore, we designed an oral anticoagulant therapy to improve the immunosuppressive tumor microenvironment and potentiate the efficacy of immunotherapy by alleviating tumor hypoxia. EXPERIMENTAL DESIGN: A novel oral anticoagulant (STP3725) was developed to consistently prevent CAT formation. Tumor perfusion and hypoxia were analyzed with or without treating STP3725 in wild-type and P selectin knockout mice. Immunosuppressive cytokines and cells were analyzed to evaluate the alteration of the tumor microenvironment. Effector lymphocyte infiltration in tumor tissue was assessed by congenic CD45.1 mouse lymphocyte transfer model with or without anticoagulant therapy. Finally, various tumor models including K-Ras mutant spontaneous cancer model were employed to validate the role of the anticoagulation therapy in enhancing the efficacy of immunotherapy. RESULTS: CAT was demonstrated to be one of the perfusion barriers, which fosters immunosuppressive microenvironment by accelerating tumor hypoxia. Consistent treatment of oral anticoagulation therapy was proved to promote tumor immunity by alleviating hypoxia. Furthermore, this resulted in decrease of both hypoxia-related immunosuppressive cytokines and myeloid-derived suppressor cells while improving the spatial distribution of effector lymphocytes and their activity. The anticancer efficacy of αPD-1 antibody was potentiated by co-treatment with STP3725, also confirmed in various tumor models including the K-Ras mutant mouse model, which is highly thrombotic. CONCLUSIONS: Collectively, these findings establish a rationale for a new and translational combination strategy of oral anticoagulation therapy with immunotherapy, especially for treating highly thrombotic cancers. The combination therapy of anticoagulants with immunotherapies can lead to substantial improvements of current approaches in the clinic.
Assuntos
Anticoagulantes/uso terapêutico , Hipóxia Celular/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Imunoterapia/métodos , Animais , Anticoagulantes/farmacologia , Humanos , Camundongos , Microambiente TumoralRESUMO
Durotaxis is the phenomena of directed cell migration driven by gradients of extracellular matrix stiffness. Durotaxis has been recently involved in the development of fibrosis by promoting the recruitment of pathological fibroblasts to areas of established fibrosis, thus amplifying the fibrotic response. Here, we describe the fabrication of mechanically patterned hydrogels that can be used to investigate molecular mechanisms controlling durotaxis of fibroblasts and other cells with mechanosensing properties. This method effectively creates a stiffness gradient of 275 Pa/µm, mimicking the natural spatial stiffness variations we observed in fibrotic tissues from mouse models of fibrosis and human fibrotic diseases.
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Técnicas de Cultura de Células/métodos , Matriz Extracelular/patologia , Fibroblastos/citologia , Fibrose Pulmonar Idiopática/patologia , Animais , Adesão Celular , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Módulo de Elasticidade , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Hidrogéis , Fibrose Pulmonar Idiopática/metabolismo , CamundongosRESUMO
ROCK, one of the downstream regulators of Rho, controls actomyosin cytoskeleton organization, stress fiber formation, smooth muscle contraction, and cell migration. ROCK plays an important role in the pathologies of cerebral and coronary vasospasm, hypertension, cancer, and arteriosclerosis. Pharmacological-induced systemic inhibition of ROCK affects both the pathological and physiological functions of Rho-kinase, resulting in hypotension, increased heart rate, decreased lymphocyte count, and eventually cardiovascular collapse. To overcome the adverse effects of systemic ROCK inhibition, we developed a bioreductive prodrug of a ROCK inhibitor, fasudil, that functions selectively under hypoxic conditions. By masking fasudil's active site with a bioreductive 4-nitrobenzyl group, we synthesized a prodrug of fasudil that is inactive in normoxia. Reduction of the protecting group initiated by hypoxia reveals an electron-donating substituent that leads to fragmentation of the parent molecule. Under normoxia the fasudil prodrug displayed significantly reduced activity against ROCK compared to its parent compound, but under severe hypoxia the prodrug was highly effective in suppressing ROCK activity. Under hypoxia the prodrug elicited an antiproliferative effect on disease-afflicted pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. The prodrug displayed a long plasma half-life, remained inactive in the blood, and produced no drop in systemic blood pressure when compared with fasudil-treated controls. Due to its selective nature, our hypoxia-activated fasudil prodrug could be used to treat diseases where tissue-hypoxia or hypoxic cells are the pathological basis of the disease.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Hipóxia , Pró-Fármacos , Inibidores de Proteínas Quinases , Quinases Associadas a rho , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/efeitos adversos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Células Endoteliais , Humanos , Hipóxia/tratamento farmacológico , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacologia , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Treatments promoting post-stroke functional recovery continue to be an unmet therapeutic problem with physical rehabilitation being the most reproduced intervention in preclinical and clinical studies. Unfortunately, physiotherapy is typically effective at high intensity and early after stroke - requirements that are hardly attainable by stroke survivors. The aim of this study was to directly evaluate and compare the dose-dependent effect of delayed physical rehabilitation (daily 5â¯h or overnight voluntary wheel running; initiated on post-stroke day 7 and continuing through day 21) on recovery of motor function in the mouse photothrombotic model of ischemic stroke and correlate it with angiogenic potential of the brain. Our observations indicate that overnight but not 5â¯h access to running wheels facilitates recovery of motor function in mice in grid-walking test. Western blotting and immunofluorescence microscopy experiments evaluating the expression of angiogenesis-associated proteins VEGFR2, doppel and PDGFRß in the peri-infarct and corresponding contralateral motor cortices indicate substantial upregulation of these proteins (≥2-fold) in the infarct core and surrounding cerebral cortex in the overnight running mice on post-stroke day 21. These findings indicate that there is a dose-dependent relationship between the extent of voluntary exercise, motor recovery and expression of angiogenesis-associated proteins in this expert-recommended mouse ischemic stroke model. Notably, our observations also point out to enhanced angiogenesis and presence of pericytes within the infarct core region during the chronic phase of stroke, suggesting a potential contribution of this tissue area in the mechanisms governing post-stroke functional recovery.
Assuntos
Proteínas Angiogênicas , Condicionamento Físico Animal , Acidente Vascular Cerebral , Animais , Modelos Animais de Doenças , Camundongos , Atividade Motora , Recuperação de Função Fisiológica , Regulação para CimaRESUMO
Currently used animal and cellular models for pulmonary arterial hypertension (PAH) only partially recapitulate its pathophysiology in humans and are thus inadequate in reproducing the hallmarks of the disease, inconsistent in portraying the sex-disparity, and unyielding to combinatorial study designs. Here we sought to deploy the ingenuity of microengineering in developing and validating a tissue chip model for human PAH. We designed and fabricated a microfluidic device to emulate the luminal, intimal, medial, adventitial, and perivascular layers of a pulmonary artery. By growing three types of pulmonary arterial cells (PACs)-endothelial, smooth muscle, and adventitial cells, we recreated the PAH pathophysiology on the device. Diseased (PAH) PACs, when grown on the chips, moved of out their designated layers and created phenomena similar to the major pathologies of human PAH: intimal thickening, muscularization, and arterial remodeling and show an endothelial to mesenchymal transition. Flow-induced stress caused control cells, grown on the chips, to undergo morphological changes and elicit arterial remodeling. Our data also suggest that the newly developed chips can be used to elucidate the sex disparity in PAH and to study the therapeutic efficacy of existing and investigational anti-PAH drugs. We believe this miniaturized device can be deployed for testing various prevailing and new hypotheses regarding the pathobiology and drug therapy in human PAH.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Modelos Animais de Doenças , Humanos , Dispositivos Lab-On-A-Chip , Artéria PulmonarRESUMO
Opportunistic fungal infections are responsible for over 1.5 million deaths per year. This has created a need for highly effective antifungal medication to be as potent as possible. In this study, we improved the efficacy of a common over the counter (OTC) antifungal skin medication, miconazole, by encapsulating nano-molecules of the drug in cholesterol/sodium oleate nano-vesicles. These nano-vesicles were characterized to optimize their size, zeta potential, polydispersity index and encapsulation efficiency. Furthermore, these nano-vesicles were compared to a conventional miconazole-based commercially available cream to determine potential improvements via permeation through the stratum corneum, cytotoxicity, and antifungal capabilities. Our results found that the vesicle size was within the nano range (~300 nm), with moderate polydispersity and stability. When compared with the commercially available cream, Actavis, as well as free miconazole, the miconazole nano-vesicle formulation displayed enhanced fungal inhibition by a factor of three or more when compared to free miconazole. Furthermore, with smaller nanoparticle (NP) sizes, higher percentages of miconazole may be delivered, further enhancing the efficacy of miconazole's antifungal capability. Cytotoxicity studies conducted with human dermal fibroblast cells confirm its biosafety and biocompatibility, as cell survival rate was observed to be twofold higher in nano-vesicle formulation than free miconazole. This formulation has the potential to treat fungal infections through increasing the retention time in the skin, improving the treatment approach, and by enhancing the efficacy via the use of nano-vesicles.
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Peroxisome-proliferator-activated-receptor-gamma (PPAR-γ) is implicated, in some capacity, in the pathogenesis of pulmonary arterial hypertension (PAH). Rosiglitazone, an oral antidiabetic and PPAR-γ agonist, has the potential to dilate pulmonary arteries and to attenuate arterial remodeling in PAH. Here, we sought to test the hypothesis that rosiglitazone can be repurposed as inhaled formulation for the treatment of PAH. We have tested this conjecture by preparing and optimizing poly(lactic-co-glycolic) acid (PLGA) based particles of rosiglitazone, assessing the drug particles for pulmonary absorption, investigating the efficacy of the plain versus particulate drug formulation in improving the respiratory hemodynamics in PAH animals, and finally studying the effect of the drug in regulating the molecular markers associated with PAH pathogenesis. The optimized particles were slightly porous and spherical, and released 87.9%⯱â¯6.7% of the drug in 24â¯h. The elimination half-life of the drug formulated in PLGA particles was 2.5-fold greater than that of the plain drug administered via the same route at the same dose. The optimized formulation, given via the pulmonary route, produced pulmonary selective vasodilation in PAH animals, but oral rosiglitazone had no effect in pulmonary hemodynamics. Rosiglitazone ameliorates the pathogenesis of PAH by balancing the molecular regulators involved in the vasoconstriction and vasodilation of human pulmonary arterial smooth muscle cells. All in all, data generated using intact animal and cellular models point to the conclusion that PLGA particles of an antidiabetic drug can be used for the treatment of a different disease, PAH.
Assuntos
Reposicionamento de Medicamentos/métodos , Hipertensão Pulmonar Primária Familiar/tratamento farmacológico , Hipoglicemiantes/farmacologia , PPAR gama/agonistas , Rosiglitazona/farmacologia , Administração por Inalação , Animais , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/uso terapêutico , Masculino , Modelos Animais , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Porosidade , Artéria Pulmonar/efeitos dos fármacos , Ratos Sprague-Dawley , Rosiglitazona/administração & dosagem , Rosiglitazona/efeitos adversos , Rosiglitazona/uso terapêutico , Resultado do TratamentoRESUMO
Three-dimensional printing (3DP), though developed for nonmedical applications and once regarded as futuristic only, has recently been deployed for the fabrication of pharmaceutical products. However, the existing feeding materials (inks and filaments) that are used for printing drug products have various shortcomings, including the lack of biocompatibility, inadequate extrudability and printability, poor drug loading, and instability. Here, we have sought to develop a filament using a single pharmaceutical polymer, with no additives, which can be multi-purposed and manipulated by computational design for the preparation of tablets with desired release and absorption patterns. As such, we have used hydroxypropyl-methylcellulose (HPMC) and diltiazem, a model drug, to prepare both drug-free and drug-impregnated filaments, and investigated their thermal and crystalline properties, studied the cytotoxicity of the filaments, designed and printed tablets with various infill densities and patterns. By alternating the drug-free and drug-impregnated filaments, we fabricated various types of tablets, studied the drug release profiles, and assessed oral absorption in rats. Both diltiazem and HPMC were stable at extrusion and printing temperatures, and the drug loading was 10% (w/w). The infill density, as well as infill patterns, influenced the drug release profile, and thus, when the infill density was increased to 100%, the percentage of drug released dramatically declined. Tablets with alternating drug-free and drug-loaded layers showed delayed and intermittent drug release, depending on when the drug-loaded layers encountered the dissolution media. Importantly, the oral absorption patterns accurately reproduced the drug release profiles and showed immediate, extended, delayed and episodic absorption of the drug from the rat gastrointestinal tract (GIT). Overall, we have demonstrated here that filaments for 3D printers can be prepared from a pharmaceutical polymer with no additives, and the novel computational design allows for fabricating tablets with the capability of producing distinct absorption patterns after oral administration.
Assuntos
Portadores de Fármacos/administração & dosagem , Derivados da Hipromelose/administração & dosagem , Impressão Tridimensional , Animais , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Diltiazem/administração & dosagem , Diltiazem/sangue , Diltiazem/química , Diltiazem/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Liberação Controlada de Fármacos , Mucosa Gástrica/metabolismo , Humanos , Derivados da Hipromelose/química , Derivados da Hipromelose/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , ComprimidosRESUMO
The use of thrombolytic therapies is limited by an increased risk of systemic hemorrhage due to lysis of hemostatic clots. We sought to develop a plasmin-based thrombolytic nanocage that efficiently dissolves the clot without causing systemic fibrinolysis or disrupting hemostatic clots. Here, we generated a double chambered short-length ferritin (sFt) construct that has an N-terminal region fused to multivalent clot targeting peptides (CLT: CNAGESSKNC) and a C-terminal end fused to a microplasmin (µPn); CLT recognizes fibrin-fibronectin complexes in clots, µPn efficiently dissolves clots, and the assembly of double chambered sFt (CLT-sFt-µPn) into nanocage structure protects the activated-µPn from its circulating inhibitors. Importantly, activated CLT-sFt-µPn thrombolytic nanocage showed a prolonged circulatory life over activated-µPn and efficiently lysed the preexisting clots in both arterial and venous thromboses models. Thus, CLT-sFt-µPn thrombolytic nanocage platform represents the prototype of a targeted clot-busting agent with high efficacy and safety over existing thrombolytic therapies.
Assuntos
Trombose Coronária/prevenção & controle , Ferritinas/química , Fibrinolisina/química , Fibrinolíticos/administração & dosagem , Nanopartículas/administração & dosagem , Fragmentos de Peptídeos/química , Terapia Trombolítica/métodos , Trombose Venosa/prevenção & controle , Animais , Trombose Coronária/patologia , Modelos Animais de Doenças , Fibrinolíticos/química , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nanopartículas/química , Ratos , Ratos Sprague-Dawley , Trombose Venosa/patologiaRESUMO
Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in in vitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Heparina de Baixo Peso Molecular/química , Linfangiogênese/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Movimento Celular , Proliferação de Células , Ácido Desoxicólico/química , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Metástase Linfática , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neovascularização Patológica/tratamento farmacológicoRESUMO
Islet transplantation is as effective as but also less immunogenic than pancreas transplantation for the treatment of type 1 diabetes mellitus. However, as the complete elimination of immunogenicity still remains a major obstacle in islet transplantation, layer-by-layer encapsulation (LbL) of pancreatic islets using biocompatible polymers offers a rational approach to reducing host immune response towards transplanted islets. We investigated the effect of LbL of non-human primate (NHP) islets on reducing immunogenicity as a preclinical model since NHPs have close phylogenetic and immunological relationship with humans. LbL with three-layers of polyethylene glycol (PEG) molecules (SH-6-arm-PEG-NHS, 6-arm-PEG-catechol and linear PEG-SH) showed a uniform nano-shielding on islets without the loss of viability or function of islets. An immunosuppressive drug protocol was also combined to improve the survival rate of the transplanted islets in vivo. A xenorecipient (C57BL/6 mice) of LbL islet transplanted along with our immunosuppressive drug protocol showed 100% survival rate for 150days after transplantation. On the other hand, naked islet recipients showed poor survival time of 5.5±1.4days without drugs and 77.5±42days with the drug protocol. Immunohistochemistry of the transplanted grafts and serum cytokine concentration demonstrated less immunogenicity in the LbL islet transplanted recipients compared with the naked islet ones.
Assuntos
Diabetes Mellitus Experimental/terapia , Imunossupressores/uso terapêutico , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Polietilenoglicóis/química , Animais , Catecóis/química , Separação Celular , Células Imobilizadas/citologia , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante HeterólogoRESUMO
Heparin and low molecular weight heparins (LMWHs) have been the drug of choice for the treatment or the prevention of thromboembolic disease. Different methods are employed to prepare the LMWHs that are clinically approved for the market currently. In particular, enoxaparin, which has a reducing sugar moiety at the end-site of polysaccharide, is prepared by alkaline depolymerization. Focusing on this end-site-specific activity of LMWHs, we conjugated the tetraoligomer of deoxycholic acid (TetraDOCA; TD) at the end-site of enoxaparin via nonenzymatic glycosylation reaction. The end-site-specific conjugation is important for polysaccharide drug development because of the heterogeneity of polysaccharides. This study also showed that orally active enoxaparin and tetraDOCA conjugate (EnoxaTD) had therapeutic effect on deep vein thrombosis (DVT) without bleeding in animal models. Considering the importance of end-specific conjugation, these results suggest that EnoxaTD could be a drug candidate for oral heparin development.
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Ácidos Cólicos/farmacologia , Sistemas de Liberação de Medicamentos , Enoxaparina/farmacologia , Trombose Venosa/tratamento farmacológico , Administração Oral , Animais , Sobrevivência Celular/efeitos dos fármacos , Ácidos Cólicos/administração & dosagem , Ácidos Cólicos/química , Cães , Relação Dose-Resposta a Droga , Enoxaparina/administração & dosagem , Enoxaparina/química , Glicosilação , Células Madin Darby de Rim Canino/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Príons/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Sistemas de Liberação de Medicamentos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Príons/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-ß1 (TGF-ß1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-ß1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-ß1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-ß1 and CXCL12 (TGF-ß1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-ß1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-ß1 and CXCL12 with LHTD4 were 0.85 and 0.019 µM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-ß1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-ß1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-ß1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-ß1 and CXCL12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models, the treatment with LHTD4 (5 mg/kg daily, p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-ß1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity.