RESUMEN
Integrins are heterodimers composed of two subunits, α(120-185kD) and ß (90-110kD), which mediate the connection between cells and their external environment, such as extracellular matrix (ECM), and play an important role in the regulation of cell shape, proliferation and migration. Herein, we identified a potent anti-tumor migration peptide Accutin from crude venom of Agkistrodon acutus using an A549 3D tumor sphere model, and simulation tools and RNA sequencing were performed to reveal the mechanism of Accutin. Accutin is a disintegrin and docking, molecular dynamics simulations and ITC assay indicate that the RGD motif in the Accutin sequence can stably bind to integrins α5ß1. 9.22 nM Accutin can significantly inhibit the migration and invasion of lung cancer cell lines. Transcriptome analysis indicated that many genes are involved in tumor cell adhesion-related biological processes. Several pathways, like the "mTOR signaling pathway", "TGF-ß signaling pathway", and "Focal adhesion" were enriched. Interestingly, pathways involved in "N-Glycan biosynthesis" etc. were significantly inhibited. These transcriptomics data suggested that the molecular basis of Accutin-mediated inhibition of cancer cell migration may be by inhibiting N-glycosylation of integrin, then inhibiting signaling pathways such as PI3K/AKT/mTOR and TGFß/smad. Western blotting analysis further confirmed that Accutin could suppress migration via down-regulating the phosphorylation of FAK and AKT and inhibiting EMT (epithelial-mesenchymal transition). Taken together, as a disintegrin with high efficiency, Accutin may be a potential precursor of a therapeutic agent for the treatment of lung cancer migration.
Asunto(s)
Movimiento Celular , Transición Epitelial-Mesenquimal , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Humanos , Movimiento Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células A549 , Quinasa 1 de Adhesión Focal/metabolismo , Desintegrinas/farmacología , Desintegrinas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbß3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.
Asunto(s)
Desintegrinas , Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Animales , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/aislamiento & purificación , Desintegrinas/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Ratones , Viperidae/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Línea Celular Tumoral , Expresión Génica , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Venenos de Crotálidos/química , Venenos de Crotálidos/genética , Crotalinae , Serpientes VenenosasRESUMEN
FS145, a protein containing a WGD motif, was previously described from the salivary transcriptome of the flea Xenopsylla cheopis. Nevertheless, its biological function and complete structure are still uncertain. Herein, FS145 was confirmed to adopt a common αßß structure with the WGD motif exposed on its surface and located right at the top of a loop composed of residues 72-81. Furthermore, FS145 dose-dependently inhibited the proliferation, adhesion, migration, and tube formation of HUVECs by not only binding to integrin αvß3 but also by subsequently inactivating the FAK/Src/MAPK pathway along with the reduction of the expression of MMP-2, MMP-9, VEGFA, bFGF, Ang2, Tie2, HIF-1α, and FAK. Moreover, FS145 also inhibited aortic vessel sprout and showed strong anti-angiogenic activities as assessed ex vivo, by employing the rat aortic ring assay, chick embryo chorioallantoic membrane, and zebrafish embryo models. Altogether, our results suggest that FS145 suppresses angiogenesis ex vivo and in vitro by blocking integrin αvß3. The current study reveals the first anti-angiogenesis disintegrin with WGD motif from invertebrates and provides a beneficial pharmacological activity to inhibit abnormal angiogenesis.