Qingyu Lang

The dietary flavonoid luteolin inhibits Aurora B kinase activity and blocks proliferation of cancer cells

In human, Aurora B is a chromosomal passenger protein that induces phosphorylation of histone and involves in spindle checkpoint and cytokinesis. Aberrant expression of Aurora B has been shown to correlate with genetic instability and carcinogenesis. In the past, Aurora B has been validated as a drug target by several studies. Here we report that the dietary flavonoid luteolin could inhibit recombinant Aurora B in radiometric activity assay (IC(50)=0.357 μM) and bind to Aurora B with a high affinity (K(D)=5.85 μM) measured by Biacore 3000. Dose-dependent down-regulation of phosphorylation on Ser10 of histone H3 was also observed in cancer cell lines after 24-h treatment, indicating that endogenous Aurora B activity was inhibited by luteolin. Furthermore, we evaluated the effects of luteolin on the survival of a panel of 23 cell lines, and found that luteolin blocked growth of HeLa cells and SW620 cells in an 8-day cell proliferation assay as well as in colony formation assay. Thus, we identified Aurora B as a novel direct target of luteolin, and our results demonstrated that targeting Aurora B by natural products may be a feasible strategy to develop low toxic anticancer agents.

In vitro and in vivo characterization of a benzofuran derivative, a potential anticancer agent, as a novel Aurora B kinase inhibitor.

Aurora B is a serine/threonine kinase that has a key role in mitosis and is overexpressed in cancer cells. Aberrations in Aurora B are highly correlated with tumorigenesis and cancer development, so many studies have focused on the development of Aurora B kinase inhibitors. Based on one of our previous high-throughput screening studies, we identified lead compound S6, a small-molecule benzofuran derivative that binds Aurora B and inhibits its kinase activity in vitro. S6 also displayed high selectivity for Aurora B inhibition. The cytotoxicity of S6 was assessed against a panel of 21 cancer cell lines. The cervical cancer cell line HeLa, liver cancer cell line HepG2 and colon cancer cell line SW620 were the most sensitive to S6 treatment. We found that S6 decreased the proliferation and colony formation of these three cell lines and elevated their percentages of cells in the G2/M phase of the cell cycle. S6 also inhibited phospho-histone H3 on Ser 10, a natural biomarker of endogenous Aurora B activity. The growth suppression of liver cancer QGY-7401 xenograft tumors was observed in nude mice after S6 administration, and this effect was accompanied by the in vivo inhibition of phospho-histone H3 (Ser 10). Taken together, we conclude that targeting Aurora B with compound S6 may be a novel strategy for cancer treatment, and additional studies are warranted.

Identification and characterization of human LYPD6, a new member of the Ly-6 superfamily

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 3,501 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 AP1.

3-Hydroxyflavone inhibits endogenous Aurora B and induces growth inhibition of cancer cell line

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.

A thienopyrimidine derivative induces growth inhibition and apoptosis in human cancer cell lines via inhibiting Aurora B kinase activity.

Aurora kinases play a key role in the regulation of mitosis and have been regarded as promising targets of cancer therapy. In this paper we describe a thienopyrimidine derivative (S7), a novel potent ATP-competitive hit inhibitor of Aurora B kinase screened through a HTS system, with the IC50 141.12 nM in the biochemical kinase activity assay. Human tumor cells treated with S7 showed dose-dependent inhibition of auto-phosphorylation of Aurora B on Thr232 and another widely-used marker specific for Aurora B kinase, the phosphorylation of Histone H3 (Ser 10), demonstrating endogenous Aurora B kinase activity were inhibited at cellular level. Moreover, S7 treatment induced proliferation inhibition, colony formation inhibition and apoptosis of human tumor cell lines in a dose- and time-dependent manner.