RESUMO
Angiogenesis is an important hallmark of cancer serving a key role in tumor growth and metastasis. Therefore, tumor angiogenesis has become an attractive target for development of novel drug therapies. An increased amount of antiangiogenic compounds is currently in preclinical and clinical development for personalized therapies. However, resistance to current angiogenesis inhibitors is emerging, indicating that there is a need to identify novel antiangiogenic agents. In the last decade, the field of microRNA biology has exploded revealing unsuspected functions in tumor angiogenesis. These small noncoding RNAs, which have been dubbed as angiomiRs, may target regulatory molecules driving angiogenesis, such as cytokines, metalloproteinases and growth factors, including vascular endothelial growth factor, plateletderived growth factor, fibroblast growth factor, epidermal growth factor, hypoxia inducible factor1, as well as mitogenactivated protein kinase, phosphoinositide 3kinase and transforming growth factor signaling pathways. The present review discusses the current progress towards understanding the functions of miRNAs in tumor angiogenesis regulation in diverse types of human cancer. Furthermore, the potential clinical application of angiomiRs towards antiangiogenic tumor therapy was explored.
Assuntos
Inibidores da Angiogênese/uso terapêutico , MicroRNAs/uso terapêutico , Neoplasias/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/genética , Animais , Feminino , Humanos , Camundongos , MicroRNAs/genética , Terapia de Alvo Molecular , Neovascularização Patológica/genética , Microambiente Tumoral/efeitos dos fármacosRESUMO
Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.
Assuntos
Inibidores da Angiogênese/genética , Furina/genética , Neovascularização Patológica/genética , Mutação Puntual/genética , Semaforinas/genética , Inibidores da Angiogênese/análise , Linhagem Celular , Furina/análise , Células Endoteliais da Veia Umbilical Humana , Humanos , Plasmídeos , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Semaforinas/análise , Fatores de Tempo , TransfecçãoRESUMO
Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.
Assuntos
Humanos , Mutação Puntual/genética , Inibidores da Angiogênese/genética , Semaforinas/genética , Furina/genética , Neovascularização Patológica/genética , Plasmídeos , Valores de Referência , Fatores de Tempo , Transfecção , Linhagem Celular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores da Angiogênese/análise , Semaforinas/análise , Furina/análise , Células Endoteliais da Veia Umbilical HumanaRESUMO
Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.
Assuntos
Inibidores da Angiogênese , Células Endoteliais/metabolismo , Biblioteca de Peptídeos , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/farmacologia , Animais , Células Endoteliais/citologia , Humanos , Camundongos , Domínios Proteicos , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Angiogenesis has been considered an important target for cancer therapy. The inhibition of angiogenesis represents a promising strategy for anti-cancer treatment, tumor growth inhibition, and metastasis. Vasostatin 30 (Vs30), and the 14.1 kDa vasoinhibin (Vi-II-14.1) are two peptides with remarkable anti-tumor and anti-angiogenic effect. The aim of this study was to produce a novel fusion protein between Vs30 and Vi-II-14.1, denominated VS_VI, to obtain a new protein with higher biological activity. The protein fusion genes were cloned into a T7 promoter-based vector, expressed in Escherichia coli BL21-SI and purified by affinity column chromatography. In vitro assays showed that the recombinant fusion protein inhibited rat coronary endothelial cell proliferation at 65.5 % at 10 nM, whereas recombinant Vs30 and Vi-II-14.1 inhibited at 33 and 50.5 % respectively, at the same concentration. The results showed that VS_VI is significantly more active than the Vs30 and Vi-II-14.1 separately. In addition, a practical classification of the vasoinhibins based on the peptide origin and theoretical molecular weight is proposed. This is the first study to produce a new fusion protein derived from Vs30 and Vi-II-14.1, both of them proposed as promising therapeutic agents.
Assuntos
Inibidores da Angiogênese/farmacologia , Calreticulina/farmacologia , Células Endoteliais/efeitos dos fármacos , Prolactina/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Calreticulina/química , Calreticulina/genética , Calreticulina/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Prolactina/química , Prolactina/genética , Prolactina/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 µl; each 48 h) inhibited VEGF-A-induced (10 ng/10 µl; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas e Peptídeos Salivares/farmacologia , Inibidores de Serina Proteinase/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Células Endoteliais/metabolismo , Inibidores do Fator Xa , Ixodidae/metabolismo , Masculino , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Tela Subcutânea/efeitos dos fármacos , Tela Subcutânea/metabolismoRESUMO
Disintegrins and disintegrins-like proteins are able to inhibit platelet aggregation and integrin-mediated cell adhesion. The aim of this study was to produce one disintegrin-like cloned from Bothrops leucurus venom gland and to characterize it regarding biological activity. The recombinant protein was purified by one step procedure involving anion-exchange chromatography (DEAE-cellulose) and presented a molecular mass of 10.4 kDa. The purified protein was able to inhibit platelet aggregation induced by collagen (IC50 = 0.65 µM) and to inhibit growth of Ehrlich tumor implanted in mice by more than 50% after 7 days administration of 10 µg/day. No effects were observed upon adenosine 5'-diphosphate (ADP)-and arachidonic acid (AA)-induced platelet aggregation. The recombinant protein was recognized by an antibody specific for jararhagin one metalloproteinase isolated from Bothrops jararaca venom, and therefore it was named leucurogin. Anti-angiogenesis effect of leucurogin was evaluated by the sponge implant model. After 7 days administration leucurogin inhibited, in a dose dependent way, the vascularization process in the sponge. Leucurogin represents a new biotechnological tool to understand biological processes where disintegrins-like are involved and may help to characterize integrins that can be involved in development and progression of malignant cells.
Assuntos
Bothrops/metabolismo , Carcinoma de Ehrlich/tratamento farmacológico , Desintegrinas/farmacologia , Proteínas Recombinantes/farmacologia , Sequência de Aminoácidos , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Bothrops/genética , Clonagem Molecular , Venenos de Crotalídeos , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/isolamento & purificação , Masculino , Metaloendopeptidases , Camundongos , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Veneno de Bothrops jararacaRESUMO
Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his)6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 microg/mL while the one for secreted (his)6-met-endostatin was about 114 microg/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS-PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his)6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF).
Assuntos
Inibidores da Angiogênese/metabolismo , Endostatinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Inibidores da Angiogênese/genética , Animais , Células CHO , Divisão Celular/fisiologia , Cricetinae , Endostatinas/química , Endostatinas/genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Camundongos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/genética , Tetra-Hidrofolato Desidrogenase/genética , TransfecçãoRESUMO
We have performed association studies between a novel coding single nucleotide polymorphism (D104N) in endostatin, one of the most potent inhibitors of angiogenesis, and prostate cancer. We observed that heterozygous N104 individuals have a 2.5 times increased chance of developing prostate cancer as compared with homozygous D104 subjects (odds ratio, 2.4; 95% confidence interval, 1.4-4.16). Modeling of the endostatin mutant showed that the N104 protein is stable. These results together with the observation that residue 104 is evolutionary conserved lead us to propose that: (a) the DNA segment containing this residue might contain a novel interaction site to a yet unknown receptor; and (b) the presence of N104 impairs the function of endostatin.
Assuntos
Adenocarcinoma/genética , Inibidores da Angiogênese/genética , Colágeno/genética , Fragmentos de Peptídeos/genética , Polimorfismo Genético , Neoplasias da Próstata/genética , Idoso , Inibidores da Angiogênese/química , Inibidores da Angiogênese/fisiologia , Colágeno/química , Colágeno/fisiologia , Endostatinas , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Knobloch syndrome (KS) is an autosomal recessive disorder defined by the occurrence of high myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities and occipital encephalocele. The KS causative gene had been assigned to a 4.3 cM interval at 21q22.3 by linkage analysis of a large consanguineous Brazilian family. We reconstructed the haplotypes of this family with ten additional markers (five were novel) and narrowed the candidate interval to a region of <245 kb, which contains 24 expressed sequence tags, the KIAA0958 gene and the 5' end of the COL18A1 gene. We identified a homozygous mutation at the AG consensus acceptor splice site of COL18A1 intron 1 exclusively among the 12 KS patients, which was not found among 140 control chromosomes. This mutation predicts the creation of a stop codon in exon 4 and therefore the truncation of the alpha1(XVIII) collagen short form, which was expressed in human adult retina. These findings provide evidence that KS is caused by mutations in COL18A1 which, therefore, has a major role in determining the retinal structure as well as in the closure of the neural tube. Therefore, we show for the first time that the absence of a collagen isoform impairs embryonic cell proliferation and/or migration as a primary or secondary effect.