Inhibitory Effect of Synthetic Flavone Derivatives on Pan-Aurora Kinases: Induction of G2/M Cell-Cycle Arrest and Apoptosis in HCT116 Human Colon Cancer Cells

Abstract

Members of the aurora kinase family are Ser/Thr kinases involved in regulating mitosis. Multiple promising clinical trials to target aurora kinases are in development. To discover flavones showing growth inhibitory effects on cancer cells, 36 flavone derivatives were prepared, and their cytotoxicity was measured using a long-term clonogenic survival assay. Their half-maximal growth inhibitory effects against HCT116 human colon cancer cells were observed at the sub-micromolar level. Pharmacophores were derived based on three-dimensional quantitative structure⁻activity calculations. Because plant-derived flavones inhibit aurora kinase B, we selected 5-methoxy-2-(2-methoxynaphthalen-1-yl)-4H-chromen-4-one (derivative 31), which showed the best half-maximal cell growth inhibitory effect, and tested whether it can inhibit aurora kinases in HCT116 colon cancer cells. We found that derivative 31 inhibited the phosphorylation of aurora kinases A, aurora kinases B and aurora kinases C, suggesting that derivative 31 is a potential pan-aurora kinase inhibitor. The results of our analysis of the binding modes between derivative 31 and aurora A and aurora B kinases using in-silico docking were consistent with the pharmacophores proposed in this study.

Keywords: apoptosis; aurora kinases; clonogenicity; colon cancer; flavones; in-silico docking; quantitative structure–activity relationship.

Figure 1

Effects of flavone derivatives on the inhibition of clonogenicity of HCT116 colon cancer cells. Cells were treated with derivative compounds at 0, 5, 10, 20 and 40 μM (A) or at 0, 0.1, 0.5, 1 and 5 μM (B). The dashed lines show the enlarged images.

Figure 2

Half-maximal cell growth inhibitory concentration (GI₅₀) values in Table 1.

Figure 3

Pharmacophores derived based on the CoMFA and CoMSIA models.

Figure 4

Effect of derivative 31 on inhibition of aurora kinases. HCT116 cells were serum-starved for 24 h in media containing 0.5% FBS, and treated with different concentrations of derivative 31 (0, 0.1, 1, 5 or 10 μM) for 3 h (A) or 5 μM derivative 31 for different times (1. 0.5, 1, 3 or 6 h) (B). Total cell lysates were immunoblotted with phospho-specific antibodies against AURKA (T288), AURKB (T232) and AURKC (T198). Anti-GAPDH antibody was used as an internal control.

Figure 5

Effect of derivative 31 on G2/M arrest and apoptosis. HCT116 cells were treated with 5 μM derivative 31 for 0, 12 and 24 h (A) or 0, 24 and 48 h (B). The cells were fixed with ethanol and stained with propidium iodide (PI). Cellular DNA contents were determined by flow cytometry. 2N, diploid; 4N, tetraploid; M1, sub-G1; M2, G1; M2, S, M4, G2/M.

Figure 6

Effect of derivative 31 on apoptosis induction. (A) HCT116 cells were treated with derivative 31 at 0, 5 and 10 μM for 48 h, and co-stained with fluorescein isothiocyanate (FITC)–annexin V and PI. Fluorescence intensity was analyzed by a NucleoCounter NC-3000 image cytometer. Scatter plots represent FITC–annexin V versus PI intensities (upper panels). Lower graphs represent populations of annexin V-positive cells. M1, annexin V-negative; M2, annexin V-positive. (B) HCT116 cells were treated with 5 μM derivative 31 for 0, 6, 12 and 48 h, and total cell lysates were immunoblotted with antibodies against cleaved-caspase-7 and poly(ADP-ribose) polymerase (PARP). Anti-GAPDH antibody was used as an internal control.

Figure 7

Image of the binding pocket of the AURKA–derivative 31 complex visualized using the PyMol program. Derivative 31 and Tyr212 are colored in green and yellow, respectively. Val147, Leu210 and Leu263 are marked in magenta color.

Figure 8

Image of the binding pocket of the AURKB–derivative 31 complex visualized using the PyMol program. Derivative 31 and Tyr156 are colored in green and yellow, respectively. Leu83, Phe88, Ala157 and Leu207 are marked in magenta color. Glu161 is marked in cyan color.