The Aurora kinases inhibitor MK0457 inhibits cell growth and induces apoptosis of the testicular germ cell cancer-derived cells NT2-D1

The members of the Aurora kinases family, Aurora-A, -B and -C are serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis, and alterations in their expression have been associated with malignant cell transformation, genomic instability and tumor progression. Deregulation of the expression of the Aurora kinases has been shown to occur also in testicular germ cell tumors (TGCT) identifying them as putative anti-cancer therapeutic targets. In the present study, we evaluated the in vitro effects of the Aurora kinases inhibitor MK0457 on proliferation, cell cycle, ploidy, apoptosis, and tumorigenicity on the TGCT-derived cell line NT2-D1. Treatment of the cells with MK0457 inhibited proliferation in a time- and dose-dependent manner, with IC50 = 17.2±3.3 nM. The MK0457 did not affect the expression of the 3 Aurora kinases, but prevented their ability to phosphorylate substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK0457-treated cells entered the mitotic process but were unable to complete it, showing soon afterwards the classical morphological features of apoptotic cells. The induction of apoptosis was confirmed by cytofluorimeter analysis, showing that the treatment with MK0457 for 6 h induced NT2-D1 cells accumulation in the G2/M phase of the cell cycle and the appearance, at the later time point of sub-G0 nuclei. The latter result was further corroborated by the detection of caspase-3 activation following 24 h treatment with the Aurora kinases inhibitor. Finally, the MK0457 completely abolished the ability of the NT2-D1 cells to form colonies in soft agar. In conclusion, the above findings demonstrate that inhibition of Aurora kinases activity is effective in reducing in vitro growth and tumorigenicity of NT2-D1 cells, and indicate its potential therapeutic value for TGCT treatment.