Aurora B Kinase Inhibitor 3 - Hydroxyflavone Anti-tumor Effect Explore Cum New Members Of The Ly-6 Gene Family Lypd6 Cloning And Characteristics

The Aurora kinases play a critical role in mitosis and have been suggested as promising targets for cancer therapy due to their frequent overexpression in a variety of tumors. Compared with established inhibitors of cell division such as the anti-tubulins, novel agents target mitotic enzymes and show similar efficacy but with fewer side effects. Several small-molecule inhibitors of Aurora kinases have been developed as anticancer agents, some of which have progressed to early clinical evaluation. Here we identified 3-hydroxyflavone as a novel Aurora B inhibitor through high throughput screening.3-hydroxyflavone showed potent inhibition to Aurora B with the IC50 on a nanomolar basis in the enzyme-based kinase activity assay. In the cell-based western blotting analysis,3-hydroxyflavone dramatically decreased the phosphorylation level of Histone H3 on the site of serine 10, demonstrating the potent endogenous Aurora B activity inhibition in cell level. The followed cell image analysis provided the consist result. To make it clear whether 3-hydroxyflavone inhibited Aurora B by direct binding or not, SPR analysis was carried out to measure the affinity of interaction between Aurora B protein and 3-hydroxyflavone and the result proved the binding with high affinity. Usually Aurora activity suppression induced cancer cell proliferation inhibition. Colony formation and cell viability with/without treatment of 3-hydroxyflavone were measured using CCK-8. The growth suppression under 3-hydroxyflavone present, and the growth recovery after being released gave strong evidence that presence of 3-hydroxyflavone efficiently inhibited the fast growth of cancer cells.In the second part we report identification and characterization of human LYPD6. The Ly-6 protein superfamily is usually identified as a group of proteins with a LU protein domain. LU domain is about 80 amino acids long and characterized by a conserved pattern of 10 cysteine residues. Here we report the cloning and characterization of a novel human LU domain-containing gene, LYPD6, isolated from human testis cDNA library, and mapped to 2q23.1-23.2 by searching the UCSC genomic database. The LYPD6 cDNA sequence of 3501 base pairs contains an open reading frame encoding 171 amino acids. Subcellular localization of LYPD6 demonstrated that the protein was localized in the cytoplasm when overexpressed in COS-7 cells. RT-PCR analysis showed that LYPD6 was widely expressed in human tissues and the expression levels in brain and heart were relatively high. Furthermore, the subsequent analysis based on reporter gene assays suggested that overexpression of LYPD6 in HEK 293T cells was able to suppress the transcriptional activities of API.