RESUMEN
Extravasation of circulating tumor cells (CTCs) from the vasculature is a key step in cancer metastasis. CTCs bind to cell adhesion molecules (CAMs) expressed by endothelial cells (ECs) for flow arrest prior to extravasation. While a number of EC-expressed CAMs have been implicated in facilitating CTC binding, this work investigated the efficacy of inhibiting cancer cell binding to human lung microvascular ECs via antibody blocking of E-selectin using antibody-functionalized gold nanoshells (NS). The antibody-functionalized gold NS were synthesized using both directional and non-directional antibody conjugation techniques with variations in synthesis parameters (linker length, amount of passivating agents, and ratio of antibodies to NS) to gain a better understanding of these properties on the resultant hydrodynamic diameter, zeta potential, and antibody loading density. We quantified the ability of E-selectin antibody-functionalized NS to bind human lung microvascular endothelial cells (HMVEC-Ls) under non-inflamed and inflamed (TNF-α) conditions to inhibit binding of triple-negative MDA-MB-231s. E-selectin-targeted NS prepared using non-directional conjugation had higher antibody loading than those prepared via directional conjugation, resulting in the conjugates having similar overall binding to HMVEC-Ls at a given antibody concentration. E-selectin-targeted NS reduced MDA-MB-231 binding to HMVEC-Ls by up to 41% as determined using an in vitro binding assay. These results provide useful insights into the characteristics of antibody-functionalized NS prepared under different conditions while also demonstrating proof of concept that these conjugates hold potential to inhibit CTC binding to ECs, a critical step in extravasation during metastasis.
RESUMEN
The Wnt, Hedgehog, and Notch signaling pathways play a crucial role in early development and the maintenance of adult tissues. When dysregulated, these developmental signaling pathways can drive the formation and progression of cancer by facilitating cell survival, proliferation, and stem-like behavior. While this makes these pathways promising targets for therapeutic intervention, their pharmacological inhibition has been challenging due to the substantial complexity that exists within each pathway and the complicated crosstalk that occurs between the pathways. Recently, several small molecule inhibitors, ribonucleic acid (RNA) molecules, and antagonistic antibodies have been developed that can suppress these signaling pathways in vitro, but many of them face systemic delivery challenges. Nanoparticle-based delivery vehicles can overcome these challenges to enhance the performance and anti-cancer effects of these therapeutic molecules. This review summarizes the mechanisms by which the Wnt, Hedgehog, and Notch signaling pathways contribute to cancer growth, and discusses various nanoparticle formulations that have been developed to deliver small molecules, RNAs, and antibodies to cancer cells to inhibit these signaling pathways and halt tumor progression. This review also outlines some of the challenges that these nanocarriers must overcome to achieve therapeutic efficacy and clinical translation.
Asunto(s)
Sistemas de Liberación de Medicamentos , Proteínas Hedgehog/metabolismo , Nanopartículas , Neoplasias/tratamiento farmacológico , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Humanos , Nanomedicina , Neoplasias/metabolismo , Vía de Señalización WntRESUMEN
We have demonstrated the existence of human cytokine synthesis inhibitory factor (CSIF) [interleukin 10 (IL-10)]. cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BCRFI. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this assay, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.
Asunto(s)
Herpesvirus Humano 4/genética , Interleucinas/genética , Sistemas de Lectura Abierta , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-10 , Interleucinas/farmacología , Leucocitos Mononucleares/inmunología , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Proteínas Recombinantes/farmacología , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
Cytokine synthesis inhibitory factor (CSIF; interleukin-10), a product of mouse TH2 T cell clones that inhibits synthesis of cytokines by mouse TH1 T cell clones, exhibits extensive sequence similarity to an uncharacterized open reading frame in the Epstein-Barr virus BCRF1. Recombinant BCRF1 protein mimics the activity of interleukin-10, suggesting that BCRF1 may have a role in the interaction of the virus with the host's immune system.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/inmunología , Interleucinas/biosíntesis , Proteínas Virales/inmunología , Animales , Línea Celular , ADN Viral/genética , Electroforesis en Gel de Poliacrilamida , Herpesvirus Humano 4/genética , Humanos , Interleucina-10 , Células Asesinas Naturales/inmunología , Ratones , Ensayo de Radioinmunoprecipitación , Linfocitos T/inmunología , Proteínas Virales/genéticaRESUMEN
DNA amplification-restricted transcription-translation (DARTT), is based on DNA amplification by the polymerase chain reaction (PCR) and uses PCR to truncate protein-encoding DNA while adding transcriptional and translational initiation signals to the segment. The amplified DNA segments are transcribed into RNA and translated into protein in vitro and the synthesized proteins are used to define functional sites. DARTT was applied to rhesus rotavirus gene segment 4 cDNA in order to create a series of carboxyl-terminal truncations and new amino termini in the encoded VP4 capsid protein. The truncated VP4 polypeptides were tested for reaction with 11 VP4-specific neutralizing monoclonal antibodies to identify the minimum polypeptides required for antibody recognition. Monoclonal antibodies 2G4, M2, and M7, which neutralize a number of serologically distinct rotaviruses, required amino acids 247-474 of VP4 for binding. DARTT is potentially applicable to the identification of discontinuous epitopes and functional domains on a variety of proteins.
Asunto(s)
Proteínas de la Cápside , ADN Viral/genética , Amplificación de Genes , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Rotavirus/genética , Transcripción Genética , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Cápside/genética , Macaca mulatta , Datos de Secuencia Molecular , Pruebas de Neutralización , Sondas de Oligonucleótidos , Mapeo RestrictivoRESUMEN
The complete gene 4 nucleotide sequence was determined for rhesus rotavirus and each of 11 viral variants selected by neutralizing monoclonal antibodies. Gene 4 is 2362 bases in length and encodes a protein, VP3, of 776 amino acids with a calculated Mr of 86,500. A conserved trypsin cleavage site, located at amino acid 247, divides VP3 into VP8 and VP5. Neutralizing monoclonal antibodies directed at VP3 were used to select variants that escaped neutralization. Each variant contains a single gene 4 mutation that permits viral growth in the presence of the antibody. Variant mutations were identified in six distinct neutralization regions in VP8 and VP5. Five of the six neutralization regions were found in VP8. The VP8 regions were primarily associated with strain-specific or limited heterotypic rotavirus neutralization. One region was identified in VP5 by three monoclonal antibodies that neutralize a broad range of rotavirus serotypes. The VP5 neutralization region is largely hydrophobic and is similar to putative fusion sequences of Sindbis and Semliki Forest viruses.