ABSTRACT
HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited cell proliferation of multiple cancer cell lines and macrophages, and the anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.
ABSTRACT
Objective:To construct a fusion protein of extracellular domain peptide fragment of muscle specific kinase ( MuSK) and fluorescent protein mCherry ,and used as antigen in the detection of antibodies against MuSK ( MuSKAb ) in the sera of patients with myasthenia gravis ( MG).Methods:The mCherry gene was amplified by PCR from vector pRSET-B and cloned into pGEM-T Easy Vector,and furthermore, cloned into Eukaryotic expression vector pMT /BiP/V5-His ( MuSK), which contains MuSK extracellular domain 22-452 amino acid peptide fragment gene to construct the fluorescent fusion protein gene MuSK -mCherry.The recombinant vector was subsequently transfected into drosophila S 2 cells for expression.The expressed fusion proteins were verified in confocal mi-croscope ,and used as antigen in the detection of MuSKAb in sera of MG patents in fluorescence immunoprecipitation test .Results:The fluorescent fusion protein MuSK-mCherry was successfully constructed and expressed.The MuSKAb in sera of patents with MG could be detected in fluorescence immunoprecipitation test using the constructed MuSK-mCherry fusion protein as antigen.Conclusion: It is available to use the constructed fluorescent fusion protein MuSK-mCherry as antigen in fluorescence immunoprecipitation test for the detection of MuSKAb in sera of patents with MG.