RESUMO
Aberrant activation of the Wnt/ß-catenin signaling pathway is prominent in the development and metastasis of non-small cell lung cancer (NSCLC). Highly effective inhibition of this pathway highlights a therapeutic avenue against NSCLC. Moreover, ß-catenin/LEF1 interaction regulates ß-catenin nuclear transport as well as the transcriptions of the key oncogenes in Wnt/ß-catenin signaling pathway. Therefore, interruption of this interaction would be a promising therapeutic strategy for NSCLC metastasis. To date, no economical and rapid high-throughput screening (HTS) assay has been reported for the discovery of ß-catenin/LEF1 interaction inhibitors. In this study, we developed a novel fluorescence polarization (FP)-based HTS assay to identify ß-catenin/LEF1 interaction inhibitors. The FITC-LEF1 sequence, incubation time, temperature, and DMSO resistance were optimized, and then a high Z' factor of 0.77 was achieved. A pilot screening of a natural product library via this established FP screening assay identified sanguinarine analogues as potential ß-catenin/LEF1 interaction inhibitors. GST pull-down and surface plasmon resonance (SPR) assay demonstrated that ß-catenin/LEF1 interaction is a potential anticancer target of sanguinarine in vitro. This newly developed FP screening assay will be vital for the rapid discovery of novel Wnt inhibitors targeting ß-catenin/LEF1 interaction.
Assuntos
Polarização de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Fator 1 de Ligação ao Facilitador Linfoide/antagonistas & inibidores , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Benzofenantridinas/química , Benzofenantridinas/metabolismo , Benzofenantridinas/farmacologia , Ligação Competitiva/efeitos dos fármacos , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Humanos , Isoquinolinas/química , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Proteínas Wnt/antagonistas & inibidoresRESUMO
OBJECTIVE: The purpose is to construct shiga toxin2A-luteinizing hormone releasing hormone (Stx2A-LHRH) recombinant toxin which can kill cancer cells specifically and try to get a new target-binding drug. METHODS: The fragment of Stx2A-LHRH DNA amplified by polymerase chain reaction (PCR) were cloned into plasmid pET-20b(+) vector. The recombinant plasmid pET-Stx2A-LHRH was constructed successfully and identified by endonucleases digestion and sequencing analysis then it was transformed to Escherichia coli BL21 (DE3) and expressed under the induction of isopropyl-beta-D-thiogalactopyranoside. RESULTS: The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel thin layer scanning proved a special band of a molecular weight of about 38,000, accounting for 10.32% of total amount of the supernatant protein. Stx2A-LHRH recombinant toxin can kill HeLa cells clearly. CONCLUSION: Stx2A-LHRH recombinant toxin may become a choice of target-binding drug in the future.