Immune Netw.  2017 Apr;17(2):110-115. 10.4110/in.2017.17.2.110.

Characterization of the Indirubin Derivative LDD970 as a Small Molecule Aurora Kinase A Inhibitor in Human Colorectal Cancer Cells

• Ndolo KM
• Park KR
• Lee HJ
• Yoon KB
• Kim YC
• Han SY

• KMID: 2376868
• DOI: http://doi.org/10.4110/in.2017.17.2.110

Abstract

Aurora kinase A plays an essential role in mitosis including chromosome separation and cytokinesis. Aberrant expression and activity of Aurora kinase A is associated with numerous malignancies including colorectal cancer followed by poor prognosis. The aim of this study is to determine the inhibitory effects of LDD970, an indirubin derivative, on Aurora kinase A in HT29 colorectal cancer cells. In vitro kinase assay revealed that, LDD970 inhibited levels of activated Aurora kinase A (ICâ‚…â‚€=0.37 mM). The inhibitory effects of LDD970 on Aurora kinase A, autophosphorylation and phosphorylation of histone H3 (Ser10), were confirmed by immunoblot analysis. Moreover, LDD970 inhibited migration of HT29 cells and upregulated apoptosis-related protein cleaved PARP. In cell viability assay, LDD970 was observed to suppress HT29 cell growth (GIâ‚…â‚€=4.22 µM). Although further studies are required, results of the present study suggest that LDD970 provide a valuable insight into small molecule indirubin derivative for therapeutic potential in human colorectal cancer.

Keyword

Aurora kinase A; Indirubin derivative; Colorectal cancer

MeSH Terms

Aurora Kinase A*
Cell Survival
Colorectal Neoplasms*
Cytokinesis
Histones
HT29 Cells
Humans*
In Vitro Techniques
Mitosis
Phosphorylation
Phosphotransferases
Prognosis
Histones
Phosphotransferases

Figure

• 

  Figure 1 Inhibition of Aurora kinase A by LDD970. (A) Chemical structure of LDD970 compound. (B) Effect of LDD970 on the in vitro kinase activity of Aurora A. This assay was performed on purified recombinant Aurora A enzyme using HTRF method.

• 

  Figure 2 Effects of LDD970 on phosphorylation of Aurora A and histone H3 in HT29 cells. Immunoblot analysis of phosphorylated Aurora A (p-Aurora A), phosphorylated histone H3 (p-H3) on Ser10 on HT29 cells. Cells were treated with LDD970 for 2 h with indicated concentration (A) or indicated times with 1 µM and 10 µM concentration (B). Lysates were analyzed using antibodies against p-Aurora A, p-H3 on Ser10. The β-actin antibody was used as a loading control.

• 

  Figure 3 Effects of LDD970 on cell growth and apoptosis. (A) Determination of cytotoxicity activity of LDD970 on HT29 cells. Cells were treated with LDD970 for 72 h. Percentage of cell growth was calculated using 0.5% DMSO treatment as a negative control. (B) Immunoblotting analysis of PARP and cleaved PARP after 48 h of treatment of LDD970.

• 

  Figure 4 Inhibition of cell migration by LDD970. After making the wound, cells were washed and treated with the indicated concentrations of LDD970. Cells were photographed at the time scratch (Time 0, upper panel). Movement of cells into the wound was observed after 24 h (middle panel) and 48 h (lower panel) using a microscope. Cell migration was assessed by recovery of the scratch.

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