RESUMO
Molecular imprinting represents one of the most promising strategies to design artificial enzyme inhibitors. However, the study of molecularly imprinted enzyme inhibitors (MIEIs) remains at a primary stage. Advanced applications of MIEIs for cell regulation have rarely been explored. Using a solid-phase oriented imprinting strategy so as to leave the active site of the enzymes accessible, we synthesized two MIEIs that exhibit high specificity and potent inhibitory effects (inhibition constant at low nM range) towards trypsin and angiogenin. The trypsin MIEI inhibits trypsin activity, tryptic digestion-induced extracellular matrix lysis and cell membrane destruction, indicating its utility in the treatment of active trypsin-dependent cell injury. The angiogenin MIEI blocks cancer cell proliferation by suppressing the ribonuclease activity of angiogenin and decreasing the angiogenin level inside and outside HeLa cells. Our work demonstrates the versatility of MIEIs for both enzyme inhibition and cell fate manipulation, showing their great potential as therapeutic drugs in biomedicine.
Assuntos
Inibidores Enzimáticos/química , Impressão Molecular/métodos , Polímeros/química , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/metabolismo , Células HeLa , Humanos , Cinética , Nanopartículas/química , Polímeros/síntese química , Polímeros/metabolismo , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/metabolismo , Tripsina/química , Tripsina/metabolismoRESUMO
Angiogenin (Ang), is a ribonucleolytic protein that is associated with angiogenesis, the formation of blood vessels. The involvement of Ang in vascularisation makes it a potential target for the identification of compounds that have the potential to inhibit the process. The compounds may be assessed for their ability to inhibit the ribonucleolytic activity of the protein and subsequently blood vessel formation, a crucial requirement for tumor formation. We report an inhibition of the ribonucleolytic activity of Ang with the gallate containing green tea polyphenols, ECG and EGCG that exhibits an increased efficacy upon forming polyphenol-capped gold nanoparticles (ECG-AuNPs and EGCG-AuNPs). The extent of inhibition was confirmed using an agarose gel-based assay followed by fluorescence titration studies that indicated a hundred fold stronger binding of polyphenol-capped gold nanoparticles (GTP-AuNPs) compared to the bare polyphenols. Interestingly, we found a change in the mode of inhibition from a noncompetitive type to a competitive mode of inhibition in case of the GTP-AuNPs, which is in agreement with the 'n' values obtained from the fluorescence quenching studies. The effect on angiogenesis has also been assessed by the chorioallantoic membrane (CAM) assay. We find an increase in the inhibition potency of GTP-AuNPs that could find applications in the development of anti-angiogenic compounds.
Assuntos
Enzimas/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Polifenóis/química , Ribonuclease Pancreático/metabolismo , Sítios de Ligação , Ligação Competitiva , Catequina/análogos & derivados , Catequina/química , Enzimas/química , Humanos , Cinética , Simulação de Acoplamento Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/genética , Espectrometria de FluorescênciaRESUMO
Flavonoids are a class of polyphenols that possess diverse properties. The structure-activity relationship of certain flavonoids and resveratrol with ribonuclease A (RNase A) has been investigated. The selected flavonoids have a similar skeleton and the positional preferences of the phenolic moieties toward inhibition of the catalytic activity of RNase A have been studied. The results obtained for RNase A inhibition by flavonoids suggest that the planarity of the molecules is necessary for effective inhibitory potency. Agarose gel electrophoresis and precipitation assay experiments along with kinetic studies reveal Ki values for the various flavonoids in the micromolar range. Minor secondary structural changes of RNase A were observed after interaction with the flavonoids. An insight into the specific amino acid involvement in the binding of the substrate using docking studies is also presented. The dipole moment of the flavonoids that depends on the orientation of the hydroxyl groups in the molecule bears direct correlation with the inhibitory potency against RNase A. The direct association of this molecular property with enzyme inhibition can be exploited for the design and development of inhibitors of proteins.
Assuntos
Flavanonas/química , Flavonoides/química , Flavonóis/química , Quempferóis/química , Quercetina/química , Resveratrol/química , Ribonuclease Pancreático/química , Animais , Domínio Catalítico , Bovinos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Flavanonas/metabolismo , Flavonoides/metabolismo , Flavonóis/metabolismo , Quempferóis/metabolismo , Cinética , Modelos Moleculares , Pâncreas/química , Pâncreas/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Quercetina/metabolismo , Resveratrol/metabolismo , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/isolamento & purificação , Ribonuclease Pancreático/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and it is of great significance to develop an ultrasensitive, cost-effective assay for RNase A detection. Electrochemiluminescence (ECL) technology has distinctive advantages in the development of biosensors for diverse targets. However, most of the ECL biosensors require the complex process of electrode modification, which is laborious and time consuming. In this work, an immobilization-free homogeneous ECL assay was developed for the highly sensitive detection of RNase A activity for the first time. On the basis of the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), a rU-containing chimeric DNA probe is designed and labeled with Ru(bpy)32+ (act as ECL indicator). The chimeric DNA probe hardly diffuses to the surface of negatively charged indium tin oxide (ITO) electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal detected. When the RNase A is present, the chimeric DNA probe is hydrolyzed into small fragments, which contains little negative charge and can diffuse easily to the ITO electrode surface due to the decreased electrostatic repulsion. In this case, an enhanced ECL signal can be detected. Under the optimal conditions, there is a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001-0.10 ng/mL, and the detection limit is 0.2 pg/mL. In addition, the proposed ECL sensing system is also applied to detect the RNase A inhibitor, taking As3+ as an example. The proposed homogeneous ECL sensing system provides a new approach for the highly sensitive and convenient detection of RNase A as well as other ribonucleases only by redesigning a responding chimeric DNA probe.
Assuntos
Arsênio/análise , Técnicas Eletroquímicas/métodos , Inibidores Enzimáticos/análise , Medições Luminescentes/métodos , Ribonuclease Pancreático/análise , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/química , Técnicas Biossensoriais/métodos , Sondas de DNA/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Corantes Fluorescentes/química , Hidrólise , Limite de Detecção , Luminescência , Compostos Organometálicos/química , Reprodutibilidade dos Testes , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/química , Compostos de Estanho/químicaRESUMO
Adenosine and uridine 5'-tetra- and 5'-pentaphosphates were synthesized from an activated tetrametaphosphate ([PPN]2[P4O11], [PPN]2[1], PPN = bis(triphenylphosphine)iminium) and subsequently tested for inhibition of the enzymatic activity of ribonuclease A (RNase A). Reagent [PPN]2[1] reacts with unprotected uridine and adenosine in the presence of a base under anhydrous conditions to give nucleoside tetrametaphosphates. Ring opening of these intermediates with tetrabutylammonium hydroxide ([TBA][OH]) yields adenosine and uridine tetraphosphates (p4A, p4U) in 92% and 85% yields, respectively, from the starting nucleoside. Treatment of ([PPN]2[1]) with AMP or UMP yields nucleoside-monophosphate tetrametaphosphates (cp4pA, cp4pU) having limited aqueous stability. Ring opening of these ultraphosphates with [TBA][OH] yields p5A and p5U in 58% and 70% yield from AMP and UMP, respectively. We characterized inorganic and nucleoside-conjugated linear and cyclic oligophosphates as competitive inhibitors of RNase A. Increasing the chain length in both linear and cyclic inorganic oligophosphates resulted in improved binding affinity. Increasing the length of oligophosphates on the 5' position of adenosine beyond three had a deleterious effect on binding. Conversely, uridine nucleotides bearing 5' oligophosphates saw progressive increases in binding with chain length. We solved X-ray cocrystal structures of the highest affinity binders from several classes. The terminal phosphate of p5A binds in the P1 enzymic subsite and forces the oligophosphate to adopt a convoluted conformation, while the oligophosphate of p5U binds in several extended conformations, targeting multiple cationic regions of the active-site cleft.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nucleosídeos/química , Nucleosídeos/farmacologia , Ribonuclease Pancreático/antagonistas & inibidores , Animais , Domínio Catalítico/efeitos dos fármacos , Bovinos , Modelos Moleculares , Polifosfatos/química , Polifosfatos/farmacologia , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismoRESUMO
Thalidomide, a well-known immunomodulatory compound, has an anti-angiogenic activity, which may be utilized for the treatment of angiogenesis-related diseases such as hemangioendothelioma. The aim of the present study was to investigate both the antitumor role of thalidomide on hemangioendothelioma and the underlying mechanism. By using the xenograft mouse model, we found that thalidomide can inhibit the progression of hemangioendothelioma in vivo. Moreover, thalidomide shows no effect on the proliferation of hemangioendothelioma endothelial cell (EOMA), but significantly impairs the pro-angiogenic capacity of the EOMA cells in vitro. By qRT-PCR screening, we observed that the expression of angiogenin was downregulated by thalidomide treatment. We next performed tissue array analysis and found a positive correlation between angiogenin expression level and hemangioendothelioma occurrence in patients. Moreover, we confirmed that the antitumoral role of thalidomide is dependent on angiogenin expression both in vivo and in vitro. Taken together, we concluded that thalidomide can inhibit the progression of hemangioendothelioma by downregulating the expression of pro-angiogenic factor angiogenin and therefore can be used as a potent therapeutic to treat hemangioendothelioma.
Assuntos
Hemangioendotelioma/prevenção & controle , Ribonuclease Pancreático/antagonistas & inibidores , Talidomida/farmacologia , Animais , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Hemangioendotelioma/patologia , Humanos , Camundongos , Ribonuclease Pancreático/fisiologiaRESUMO
BACKGROUND: Increased activity of the receptor tyrosine kinase Tie2 has been implicated in the promotion of pathological angiogenesis. This activity is mainly mediated through angiopoietin (Ang)1- and Ang2-dependent activation of integrins by Tie2, rendering the Ang/Tie2/integrin axis an attractive putative target for cancer therapeutics. RESULTS: To target this axis, we developed single domain, non-immunoglobulin high-affinity bi-specific protein inhibitors against both Tie2 and αvß3 integrin. We have previously engineered the Ang2-binding domain of Tie2 (Ang2-BD) as a Tie2 inhibitor. Here, we engineered an exposed loop in Ang2-BD to generate variants that include an integrin-binding Arg-Gly-Asp (RGD) motif and used flow cytometry screening of a yeast-displayed Ang2-BD RGD loop library to identify the integrin antagonists. The bi-specific antagonists targeting both Tie2 and αvß3 integrin inhibited adhesion and proliferation of endothelial cells cultured together with the αvß3 integrin ligand vitronectin, as well as endothelial cell invasion and tube formation. The bi-specific reagents inhibited downstream signaling by Tie2 intracellularly in response to its agonist Ang1 more effectively than the wild-type Ang2 BD that binds Tie2 alone. CONCLUSIONS: Collectively, this study-the first to describe inhibitors targeting all the known functions resulting from Tie2/integrin αvß3 cross-talk-has created new tools for studying Tie2- and integrin αvß3-dependent molecular pathways and provides the basis for the rational and combinatorial engineering of ligand-Tie2 and ligand-integrin αvß3 receptor interactions. Given the roles of these pathways in cancer angiogenesis and metastasis, this proof of principle study paves the route to create novel Tie2/integrin αvß3-targeting proteins for clinical use as imaging and therapeutic agents.
Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização Fisiológica/genética , Receptor TIE-2/antagonistas & inibidores , Receptores de Vitronectina/genética , Ribonuclease Pancreático/antagonistas & inibidores , Inibidores da Angiogênese/química , Animais , Camundongos , Receptor TIE-2/química , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Receptores de Vitronectina/química , Receptores de Vitronectina/metabolismo , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismoRESUMO
BACKGROUND: Graphene oxide (GO) has attracted intensive interest in biological and medical fields in recent years due to its unique physical, chemical, and biological properties. In our previous work, we proved that GO could deliver small interfering RNA (siRNA) into cells and downregulate the expression of the desired gene. METHODS: This study investigated the potential of a modified GO nanocarrier for co-delivery of siRNA and doxorubicin (DOX) for enhanced cancer therapy. Fourier transform infrared spectroscopy, laser particle size analyzer, UV-visible spectroscopy, gel electrophoresis retardation, and in vitro release assay were studied. RESULTS: The results of real-time polymerase chain reaction revealed that the expression of vascular endothelial growth factor (VEGF) mRNA was decreased 46.84%±3.72% (mean ± SD). Enzyme-linked immunosorbent assay indicated that the expression of VEGF protein was down-regulated to 52.86%±1.10% (mean ± SD) in vitro. In vivo tumor growth assay GO-poly-l-lysine hydrobromide/folic acid (GPF)/DOX/siRNA exhibited gene silencing and tumor inhibition (66.95%±2.35%, mean ± SD) compared with naked siRNA (1.62%±1.47%, mean ± SD) and DOX (33.63%±5.85%, mean ± SD). GPF/DOX/siRNA exhibited no testable cytotoxicity. CONCLUSION: The results indicated that co-delivery of siRNA and DOX by GPF could be a promising application in tumor clinical therapy.
Assuntos
Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Grafite/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Ácido Fólico/química , Inativação Gênica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos Endogâmicos ICR , Nanopartículas/ultraestrutura , Neoplasias/patologia , Polilisina/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease Pancreático/antagonistas & inibidores , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND/AIMS: Patients with hepatitis B virus (HBV) infection are at a high risk of developing hepatocellular carcinoma (HCC). In this study, we aim to investigate the roles of HBV on angiogenin (ANG), as well as the effects on cell proliferation in presence of ANG down-regulation. METHODS: Serum ANG was determined by ELISA. The expression of ANG mRNA and protein in HCC cell lines with or without HBV/HBx were determined. Western blot and ELISA were conducted to determine the effects of HBV/HBx on IL-6 expression. The role of IL-6 on ANG was evaluated by IL-6 recombinant protein or IL-6 neutralizing antibody. Immunofluorescence staining was used to detect the nuclear translocation of ANG. MTT was performed to evaluate the relative inhibition ratio. RESULT: In vivo experiments showed elevation of serum ANG in patients infected with HBV. In vitro experiments showed HBV and HBx contributed to the transcription and translation of ANG. ANG expression showed increase after IL-6 stimulation, and ANG protein decreased in the presence of IL-6 blocking with its antibody. HBV promoted nuclear translocation of ANG. Inhibiting ANG expression or blocking of nuclear transfer of ANG attenuated the 45S rRNA synthesis and cell proliferation. CONCLUSION: HBV and HBx protein can increase the level of ANG through IL-6. HBV and HBx contributed to the nuclear translocation of ANG. Cell proliferation was inhibited after inhibiting the expression or nuclear transfer of ANG.
Assuntos
Carcinoma Hepatocelular/diagnóstico , Vírus da Hepatite B/fisiologia , Interleucina-6/farmacologia , Neoplasias Hepáticas/diagnóstico , Ribonuclease Pancreático/sangue , Regulação para Cima/efeitos dos fármacos , Adulto , Anticorpos/imunologia , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Células Hep G2 , Hepatite B/complicações , Hepatite B/diagnóstico , Humanos , Interleucina-6/imunologia , Interleucina-6/metabolismo , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Interferência de RNA , RNA Ribossômico/metabolismo , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Transativadores/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba2+, Co2+, Pb2+, As3+, and Cu2+ inhibited RNase A activity with IC50 values of 93.7 µM (Ba2+), 90.9 µM (Co2+), 110.6 µM (Pb2+), 171.5 µM (As3+), and 165.1 µM (Cu2+), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.
Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Corantes Fluorescentes/química , Grafite/química , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/análise , Antivirais/química , Antivirais/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Corantes Fluorescentes/metabolismo , Grafite/metabolismo , Hepatite B/tratamento farmacológico , Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/metabolismo , Humanos , Juglandaceae/química , Metais Pesados/química , Metais Pesados/farmacologia , Testes de Sensibilidade Microbiana , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Folhas de Planta/química , Ribonuclease Pancreático/metabolismo , Espectrometria de FluorescênciaRESUMO
Interaction between angiogenin and the p53 TAD2 domain in cancer cells can inhibit the function of the p53 tumor suppressor and promote cell survival. Based on a model structure using NMR and mutational analysis, positively charged 31 RRR33 and 50 KRSIK54 motifs of human angiogenin were identified as p53-binding sites that could interact with negatively charged D48/E51 and E56 residues of the p53 TAD2 domain, respectively. These results suggest that 31 RRR33 and 50 KRSIK54 motifs of human angiogenin might play a critical role in the regulation of p53-mediated apoptosis and angiogenesis in cancer cells. This study identifies potential target sites for screening angiogenin-specific inhibitors that could not only inhibit p53 binding but could also simultaneously inhibit cell binding, internalization, DNA binding, and nuclear translocation of human angiogenin.
Assuntos
Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Apoptose , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease Pancreático/química , Eletricidade Estática , Proteína Supressora de Tumor p53/químicaRESUMO
A destabilized tumor vasculature leads to limited drug delivery, hypoxia, detrimental tumor microenvironment, and even metastasis. We performed a side-by-side comparison of ABTAA (Ang2-Binding and Tie2-Activating Antibody) and ABA (Ang2-Blocking Antibody) in mice with orthotopically implanted glioma, with subcutaneously implanted Lewis lung carcinoma, and with spontaneous mammary cancer. We found that Tie2 activation induced tumor vascular normalization, leading to enhanced blood perfusion and chemotherapeutic drug delivery, markedly lessened lactate acidosis, and reduced tumor growth and metastasis. Moreover, ABTAA favorably altered the immune cell profile within tumors. Together, our findings establish that simultaneous Tie2 activation and Ang2 inhibition form a powerful therapeutic strategy to elicit a favorable tumor microenvironment and enhanced delivery of a chemotherapeutic agent into tumors.
Assuntos
Anticorpos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Glioma/tratamento farmacológico , Receptor TIE-2/metabolismo , Ribonuclease Pancreático/antagonistas & inibidores , Animais , Anticorpos/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Neoplasias da Mama/irrigação sanguínea , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Sinergismo Farmacológico , Feminino , Glioma/irrigação sanguínea , Humanos , Camundongos , Transplante de Neoplasias , Ligação Proteica/efeitos dos fármacos , Temozolomida , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacosRESUMO
Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti-Ang-2-neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti-colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.
Assuntos
Anticorpos Antineoplásicos/farmacologia , Glioblastoma , Macrófagos , Proteínas de Neoplasias , Neoplasias Experimentais , Neovascularização Patológica , Quinazolinas/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Ribonuclease Pancreático , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/metabolismoRESUMO
Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Glioblastoma/tratamento farmacológico , Macrófagos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Ribonuclease Pancreático/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteínas de Transporte Vesicular/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Macrófagos/patologia , Camundongos , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Ribonuclease Pancreático/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND/AIM: Head and neck cancers are the fifth most common cancer type worldwide, affecting more than half a million patients annually. Development of effective therapeutic drugs is, therefore, required for this type of disease. This study assessed the effects of synthetic terrein on head and neck cancer. MATERIALS AND METHODS: Synthetic terrein was prepared by using the modified Altenhach's procedure. The effect of synthetic terrein on cell proliferation of head and neck cancer cells and HUVECs was assessed. Angiogenin secretion and ribosome biogenesis were examined by ELISA and silver staining of the nucleolar organizer region. A mouse xenograft model was prepared by inoculating mice with suspensions of cells of the human head and neck cancer cell line OSC-19 subcutaneously into the dorsal region of each mouse. Ki-67, CD31 and angiogenin expression in xenografted tumors was examined by immunohistochemistry. RESULTS: Synthetic terrein inhibited the growth of various head and neck cancer cells. In addition, an in vivo experiment revealed that synthetic terrein inhibited a xenograft tumor growth in athymic mice. Immunohistochemical analysis revealed that expression of Ki-67, CD31 and ANG was down-regulated in synthetic terrein-treated tumors, compared to controls. Synthetic terrein suppressed the ANG secretion and ribosome biogenesis in cancer cells, and cell proliferation in vascular endothelial cells. CONCLUSION: The mechanism underlying the anti-tumor effects of synthetic terrein against head and neck cancer consists of the inhibition of both tumor cell proliferation and angiogenesis via the suppression of ANG production.
Assuntos
Antineoplásicos/farmacologia , Ciclopentanos/farmacologia , Neoplasias de Cabeça e Pescoço/patologia , Ribonuclease Pancreático/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Neoplasias de Cabeça e Pescoço/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Patológica/prevenção & controle , Ribonuclease Pancreático/biossínteseRESUMO
Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.
Assuntos
Infecções por Mycoplasma/patologia , Mycoplasma pulmonis/fisiologia , Ribonuclease Pancreático/antagonistas & inibidores , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Feminino , Inflamação , Linfangiogênese , Sistema Linfático/metabolismo , Sistema Linfático/patologia , Vasos Linfáticos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Mycoplasma/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Pericitos/patologia , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Ribonuclease Pancreático/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Angiopoietin-2 (ANG2/ANGPT2) is a context-dependent TIE2 receptor agonist/antagonist and proangiogenic factor. Although ANG2 neutralization improves tumor angiogenesis and growth inhibition by vascular endothelial growth factor (VEGF)-A signaling blockade, the mechanistic underpinnings of such therapeutic benefits remain poorly explored. We employed late-stage RIP1-Tag2 pancreatic neuroendocrine tumors (PNETs) and MMTV-PyMT mammary adenocarcinomas, which develop resistance to VEGF receptor 2 (VEGFR2) blockade. We found that VEGFR2 inhibition upregulated ANG2 and vascular TIE2 and enhanced infiltration by TIE2-expressing macrophages in the PNETs. Dual ANG2/VEGFR2 blockade suppressed revascularization and progression in most of the PNETs, whereas it had only minor additive effects in the mammary tumors, which did not upregulate ANG2 upon VEGFR2 inhibition. ANG2/VEGFR2 blockade did not elicit increased PNET invasion and metastasis, although it exacerbated tumor hypoxia and hematopoietic cell infiltration. These findings suggest that evasive tumor resistance to anti-VEGFA therapy may involve the adaptive enforcement of ANG2-TIE2 signaling, which can be reversed by ANG2 neutralization.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/metabolismo , Ribonuclease Pancreático/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adenocarcinoma/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/tratamento farmacológico , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/imunologia , Transdução de Sinais , Regulação para Cima , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
BACKGROUND: MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. METHODS: The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. RESULTS: The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. CONCLUSIONS: Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.
Assuntos
Biomarcadores Tumorais/sangue , Eritrócitos/metabolismo , MicroRNAs/sangue , MicroRNAs/química , Soro/metabolismo , Inibidores Enzimáticos/farmacologia , Voluntários Saudáveis , Humanos , Estabilidade de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease Pancreático/antagonistas & inibidoresRESUMO
Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi's sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG's nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV(+) PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.
Assuntos
Antineoplásicos/administração & dosagem , Framicetina/administração & dosagem , Herpesvirus Humano 8/fisiologia , Linfoma de Efusão Primária/tratamento farmacológico , Neomicina/administração & dosagem , Ribonuclease Pancreático/antagonistas & inibidores , Animais , Ascite/patologia , Peso Corporal , Linhagem Celular Tumoral , Modelos Animais de Doenças , Linfoma de Efusão Primária/patologia , Camundongos , Camundongos SCID , Baço/patologia , Análise de Sobrevida , Resultado do TratamentoRESUMO
UNLABELLED: The androgen receptor (AR) is a critical effector of prostate cancer development and progression. Androgen-dependent prostate cancer is reliant on the function of AR for growth and progression. Most castration-resistant prostate cancer (CRPC) remains dependent on AR signaling for survival and growth. Ribosomal RNA (rRNA) is essential for both androgen-dependent and castration-resistant growth of prostate cancer cells. During androgen-dependent growth of prostate cells, androgen-AR signaling leads to the accumulation of rRNA. However, the mechanism by which AR regulates rRNA transcription is unknown. Here, investigation revealed that angiogenin (ANG), a member of the secreted ribonuclease superfamily, is upregulated in prostate cancer and mediates androgen-stimulated rRNA transcription in prostate cancer cells. Upon androgen stimulation, ANG undergoes nuclear translocation in androgen-dependent prostate cancer cells, where it binds to the rDNA promoter and stimulates rRNA transcription. ANG antagonists inhibit androgen-induced rRNA transcription and cell proliferation in androgen-dependent prostate cancer cells. Interestingly, ANG also mediates androgen-independent rRNA transcription through a mechanism that involves its constitutive nuclear translocation in androgen-insensitive prostate cancer cells, resulting in a constant rRNA overproduction and thereby stimulating cell proliferation. Critically, ANG overexpression in androgen-dependent prostate cancer cells enables castration-resistant growth of otherwise androgen-dependent cells. Thus, ANG-stimulated rRNA transcription is not only an essential component for androgen-dependent growth of prostate cancer but also contributes to the transition of prostate cancer from androgen-dependent to castration-resistant growth status. IMPLICATIONS: The ability of angiogenin to regulate rRNA transcription and prostate cancer growth makes it a viable target for therapy.